Ellen de Waal

Positive control of cell division: FtsZ is recruited by SsgB during sporulation of Streptomyces

In bacteria that divide by binary fission, cell division starts with the polymerization of the tubulin homolog FtsZ at mid-cell to form a cell division scaffold (the Z ring), followed by recruitment of the other divisome components. The current view of bacterial cell division control starts from the principle of negative checkpoints that prevent incorrect Z-ring positioning. Here we provide evidence of positive control of cell division during sporulation of Streptomyces, via the direct recruitment of FtsZ by the membrane-associated divisome component SsgB. In vitro studies demonstrated that SsgB promotes the polymerization of FtsZ. The interactions are shown in vivo by time-lapse imaging and Förster resonance energy transfer and fluorescence lifetime imaging microscopy (FRET-FLIM), and are corroborated independently via two-hybrid studies. As determined by fluorescence recovery after photobleaching (FRAP), the turnover of FtsZ protofilaments increased strongly at the time of Z-ring formation. The surprising positive control of Z-ring formation by SsgB implies the evolution of an entirely new way of Z-ring control, which may be explained by the absence of a mid-cell reference point in the long multinucleoid hyphae. In turn, the localization of SsgB is mediated through the orthologous SsgA, and premature expression of the latter is sufficient to directly activate multiple Z-ring formation and hyperdivision at early stages of the Streptomyces cell cycle.

Determination of the genetic diversity of microbial communities using DGGE analysis of PCR-amplified 16S rDNA

Microbial ecosystems contain a large diversity of bacterial species. They are dominated by complex interactions between the different microorganisms, whereby each of the individual species has a specific role in the maintenance of the system. The active communities can efficiently scavenge nutrients from the environment and eliminate toxic compounds. However, not all of the species are active; most of them are dormant until environmental conditions change to favour their growth. Because of the great metabolic diversity and flexible organisation microbial ecosystems can be found nearly everywhere. Examples are the bacterial biofilms in waste water treatment reactors, on ship walls, or the microbial mats, found in hypersaline environments, tidal sediments and hot springs.

Transfer–messenger RNA controls the translation of cell‐cycle and stress proteins in Streptomyces

The transfer–messenger RNA (tmRNA)‐mediated trans‐translation mechanism is highly conserved in bacteria and functions primarily as a system for the rescue of stalled ribosomes and the removal of aberrantly produced proteins. Here, we show that in the antibiotic‐producing soil bacterium Streptomyces coelicolor, trans‐translation has a specialized role in stress management. Analysis of proteins that were carboxy‐terminally His8‐tagged by a recombinant tmRNA identified only 10 targets, including the stress proteins: DnaK heat‐shock protein 70, thiostrepton‐induced protein A, universal stress protein A, elongation factor Tu3, and the cell‐cycle control proteins DasR, SsgA, SsgF and SsgR. Although tmRNA‐tagged proteins are degraded swiftly, the translation of dnaK and dasR messenger RNAs (mRNAs) depends fully on tmRNA, whereas transcription is unaffected. The data unveil a surprisingly dedicated functionality for tmRNA, promoting the translation of the same mRNA it targets, at the expense of sacrificing the first nascent protein. In streptomycetes, tmRNA has evolved into a dedicated task force that ensures the instantaneous response to the exposure to stress.

Effects of Cavity-Forming Mutations on the Internal Dynamics of Azurin

The effects of two single-point cavity-forming mutations, F110S and I7S, on the internal dynamics of azurin from Pseudomonas aeruginosa were probed by the phosphorescence emission of Trp-48, deeply buried in the compact hydrophobic core of the macromolecule. Changes in flexibility of the protein matrix around the chromophore were monitored by the intrinsic phosphorescence lifetime (tau(0)) whereas more general effects on structural fluctuations were deduced from the phosphorescence acrylamide quenching rate constant (k(q)), which measures the diffusion of the solute through the protein fold. The results show a spectacular, 4-5 orders of magnitude, increase of k(q) emphasizing that large amplitude structural fluctuations permitting acrylamide migration to the protein core have been drastically enhanced in each azurin mutant. The large, 12-15 kcal/mol, decrease in the activation enthalpy associated to k(q) suggests that the rate enhancement is caused, rather than through a generalized increase of protein flexibility, by the elimination of an inner barrier to the diffusion process. According to tau(0) the chromophore environment is more fluid with I7S but strikingly more rigid with F110S, demonstrating that when internal cavities are formed local effects on the mobility at the mutation site are unpredictable. Both tau(0) and k(q) reveal a structure tightening role of bound Cd(2+) that correlates with the increase in stability from apo- to holo-azurin. While these alterations in internal dynamics of azurin do not seem to play a role on electron transfer through the central region, the enhanced migration of acrylamide emphasizes that cavities may be critical for the rapid diffusion of substrates to buried, solvent inaccessible sites of enzymes.

Replacement of the methionine axial ligand in cytochrome c550 by a lysine: effects on the haem electronic structure

The prosthetic group of low‐spin haem proteins is an iron porphyrin with two axial ligands, typically histidine, methionine or lysine. Determining the geometry of the axial ligands is an important step in structural characterisation, particularly in the paramagnetic oxidised forms. This work extends earlier studies of the hyperfine nuclear magnetic resonance (NMR) shifts of haem substituents in bis‐His and His–Met cytochromes to His–Lys co‐ordination in the M100K mutant of Paracoccus versutus cytochrome c 550. The electronic structure of the His–Lys haem is shown to be similar to that produced by His–cyanide co‐ordination, such that NMR can be used to determine the geometry of the His ligand.

Expression, purification and characterization of the soluble CuA domain of cytochrome c oxidase ofParacoccus versutus

The key subunit II of cytochrome c oxidase (CcO) contains a soluble binuclear copper center (CuA) domain. The Cua domain ofParacoccus versutus was cloned, expressed, purified and characterized. The gene encoding the Cua domain in pET11d vector was expressed inE. coli BL21 (DE3). The results showed that the Cua domain was expressed mostly in inclusion bodies and the Cua domain protein synthesized inE. coli cells represents approximately 10 percent of the total cellular proteins. Dissolved in urea, dialyzed and recombined with Cu+/Cu2+ and purified by the Q-sepharose fast flow anion-exchange column and Sephadex G-75 gel filtration column, the soluble purple-colored protein, which shows a single band in electrophoresis, was obtained. The UV-visible absorption spectrum of Cua domain showed that there are intense band at 478 nm and a shoulder peak at 530 nm, and two weak bands at 360 and 806 nm respectively, which can be assigned to the charge transfer and the interactions of obitals of Cu-S and Cu-Cu in the mixed-valence binuclear metal center (Cu2S2R2). The far-UV CD spectrum indicated that this domain is predominantly in β-sheet structure. The fluorescence spectra showed that its maximal excitation wavelength and maximal emission wavelength are at 280 and 345 nm, respectively.