The EpsE Flagellar Clutch Is Bifunctional and Synergizes with EPS Biosynthesis to Promote Bacillus subtilis Biofilm Formation

Many bacteria inhibit motility concomitant with the synthesis of an extracellular polysaccharide matrix and the formation of biofilm aggregates. In Bacillus subtilis biofilms, motility is inhibited by EpsE, which acts as a clutch on the flagella rotor to inhibit motility, and which is encoded within the 15 gene eps operon required for EPS production. EpsE shows sequence similarity to the glycosyltransferase family of enzymes, and we demonstrate that the conserved active site motif is required for EPS biosynthesis. We also screen for residues specifically required for either clutch or enzymatic activity and demonstrate that the two functions are genetically separable. Finally, we show that, whereas EPS synthesis activity is dominant for biofilm formation, both functions of EpsE synergize to stabilize cell aggregates and relieve selective pressure to abolish motility by genetic mutation. Thus, the transition from motility to biofilm formation may be governed by a single bifunctional enzyme

Mutational analysis of colicin E9

SIGLEAvailable from British Library Document Supply Centre-DSC:DXN008913 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Identification of essential amino acids in the <i>Sinorhizobium meliloti</i> glucosyltransferase ExoM

International audienc

The structural and energetic basis for high selectivity in a high-affinity protein-protein interaction

High-affinity, high-selectivity protein-protein interactions that are critical for cell survival present an evolutionary paradox: How does selectivity evolve when acquired mutations risk a lethal loss of high-affinity binding? A detailed understanding of selectivity in such complexes requires structural information on weak, noncognate complexes which can be difficult to obtain due to their transient and dynamic nature. Using NMR-based docking as a guide, we deployed a disulfide-trapping strategy on a noncognate complex between the colicin E9 endonuclease (E9 DNase) and immunity protein 2 (Im2), which is seven orders of magnitude weaker binding than the cognate femtomolar E9 DNase-Im9 interaction. The 1.77 A crystal structure of the E9 DNase-Im2 complex reveals an entirely noncovalent interface where the intersubunit disulfide merely supports the crystal lattice. In combination with computational alanine scanning of interfacial residues, the structure reveals that the driving force for binding is so strong that a severely unfavorable specificity contact is tolerated at the interface and as a result the complex becomes weakened through "frustration." As well as rationalizing past mutational and thermodynamic data, comparing our noncognate structure with previous cognate complexes highlights the importance of loop regions in developing selectivity and accentuates the multiple roles of buried water molecules that stabilize, ameliorate, or aggravate interfacial contacts. The study provides direct support for dual-recognition in colicin DNase-Im protein complexes and shows that weakened noncognate complexes are primed for high-affinity binding, which can be achieved by economical mutation of a limited number of residues at the interface

Clusters in an intrinsically disordered protein create a protein-binding site: The TolB-binding region of colicin E9

The 61-kDa colicin E9 protein toxin enters the cytoplasm of susceptible cells by interacting with outer membrane and periplasmic helper proteins and kills them by hydrolyzing their DNA. The membrane translocation function is located in the N-terminal domain of the colicin, with a key signal sequence being a pentapeptide region that governs the interaction with the helper protein To1B (the To1B box). Previous NMR studies [Collins et al. (2002) J. Mol. Biol. 318, 787-904; MacDonald et al. (2004), J. Biomol. NMR 30, 81-96] have shown that the N-terminal 83 residues of colicin E9, which includes the To1B box, is intrinsically disordered and contains clusters of interacting side chains. To further define the properties of this region of colicin E9, we have investigated the effects on the dynamical and To1B-binding properties of three mutations of colicin E9 that inactivate it as a toxin. The mutations were contained in a fusion protein consisting of residues 1-61 of colicin E9 connected to the N terminus of the E9 DNase by an eight-residue linking sequence. The NMR data reveals that the mutations cause major alterations to the properties of some of the clusters, consistent with some form of association between them and other more distant parts of the amino acid sequence, particularly toward the N terminus of the protein. However, N-15 T-2 measurements indicates that residues 5-13 of the fusion protein bound to the 43-kDa To1B remain as flexible as they are in the free protein. The NMR data point to considerable dynamic ordering within the intrinsically disordered translocation domain of the colicin that is important for creating the To1B-binding site. Furthermore, amino acid sequence considerations suggest that the clusters of amino acids occur because of the size and polarities of the side chains forming them influenced by the propensities of the residues within the clusters and those immediately surrounding them in sequence space to form beta turns

Supramolecular assemblies underpin turnover of outer membrane proteins in bacteria.

Gram-negative bacteria inhabit a broad range of ecological niches. For Escherichia coli, this includes river water as well as humans and animals, where it can be both a commensal and a pathogen. Intricate regulatory mechanisms ensure that bacteria have the right complement of β-barrel outer membrane proteins (OMPs) to enable adaptation to a particular habitat. Yet no mechanism is known for replacing OMPs in the outer membrane, an issue that is further confounded by the lack of an energy source and the high stability and abundance of OMPs. Here we uncover the process underpinning OMP turnover in E. coli and show it to be passive and binary in nature, in which old OMPs are displaced to the poles of growing cells as new OMPs take their place. Using fluorescent colicins as OMP-specific probes, in combination with ensemble and single-molecule fluorescence microscopy in vivo and in vitro, as well as molecular dynamics simulations, we established the mechanism for binary OMP partitioning. OMPs clustered to form ∼0.5-μm diameter islands, where their diffusion is restricted by promiscuous interactions with other OMPs. OMP islands were distributed throughout the cell and contained the Bam complex, which catalyses the insertion of OMPs in the outer membrane. However, OMP biogenesis occurred as a gradient that was highest at mid-cell but largely absent at cell poles. The cumulative effect is to push old OMP islands towards the poles of growing cells, leading to a binary distribution when cells divide. Hence, the outer membrane of a Gram-negative bacterium is a spatially and temporally organized structure, and this organization lies at the heart of how OMPs are turned over in the membrane

Identification of a novel group of putative Arabidopsis thaliana beta-(1,3)-galactosyltransferases

The original publication can be found at www.springerlink.comTo begin biochemical and molecular studies on the biosynthesis of the type II arabinogalactan chains on arabinogalactan-proteins (AGPs), we adopted a bioinformatic approach to identify and systematically characterise the putative galactosyltransferases (GalTs) responsible for synthesizing the β-(1,3)-Gal linkage from CAZy GT-family-31 from Arabidopsis thaliana. These analyses confirmed that 20 members of the GT-31 family contained domains/motifs typical of biochemically characterised β-(1,3)-GTs from mammalian systems. Microarray data confirm that members of this family are expressed throughout all tissues making them likely candidates for the assembly of the ubiquitously found AGPs. One member, At1g77810, was selected for further analysis including location studies that confirmed its presence in the Golgi and preliminary enzyme substrate specificity studies that demonstrated β-(1,3)-GalT activity. This bioinformatic/molecular study of CAZy GT-family-31 was validated by the recent report of Strasser et al. (Plant Cell 19:2278–2292, 2007) that another member of this family (At1g26810; GALT1) encodes a β-(1,3)-GalT involved in the biosynthesis of the Lewis a epitope of N-glycans in Arabidopsis thaliana.Yongmei Qu, Jack Egelund, Paul R. Gilson, Fiona Houghton, Paul A. Gleeson, Carolyn J. Schultz and Antony Baci