Processivity of peptidoglycan synthesis provides a built-in mechanism for the robustness of straight-rod cell morphology

The propagation of cell shape across generations is remarkably robust in most bacteria. Even when deformations are acquired, growing cells progressively recover their original shape once the deforming factors are eliminated. For instance, straight-rod-shaped bacteria grow curved when confined to circular microchambers, but straighten in a growth-dependent fashion when released. Bacterial cell shape is maintained by the peptidoglycan (PG) cell wall, a giant macromolecule of glycan strands that are synthesized by processive enzymes and cross-linked by peptide chains. Changes in cell geometry require modifying the PG and therefore depend directly on the molecular-scale properties of PG structure and synthesis. Using a mathematical model we quantify the straightening of curved Caulobacter crescentus cells after disruption of the cell-curving crescentin structure. We observe that cells straighten at a rate that is about half (57%) the cell growth rate. Next we show that in the absence of other effects there exists a mathematical relationship between the rate of cell straightening and the processivity of PG synthesis—the number of subunits incorporated before termination of synthesis. From the measured rate of cell straightening this relationship predicts processivity values that are in good agreement with our estimates from published data. Finally, we consider the possible role of three other mechanisms in cell straightening. We conclude that regardless of the involvement of other factors, intrinsic properties of PG processivity provide a robust mechanism for cell straightening that is hardwired to the cell wall synthesis machinery.     

Structural insight on the recognition of surface-bound opsonins by the integrin I domain of complement receptor 3

Complement receptors (CRs), expressed notably on myeloid and lymphoid cells, play an essential function in the elimination of complement-opsonized pathogens and apoptotic/necrotic cells. In addition, these receptors are crucial for the cross-talk between the innate and adaptive branches of the immune system. CR3 (also known as Mac-1, integrin α_Mβ₂, or CD11b/CD18) is expressed on all macrophages and recognizes iC3b on complement-opsonized objects, enabling their phagocytosis. We demonstrate that the C3d moiety of iC3b harbors the binding site for the CR3 αI domain, and our structure of the C3d:αI domain complex rationalizes the CR3 selectivity for iC3b. Based on extensive structural analysis, we suggest that the choice between a ligand glutamate or aspartate for coordination of a receptor metal ion-dependent adhesion site–bound metal ion is governed by the secondary structure of the ligand. Comparison of our structure to the CR2:C3d complex and the in vitro formation of a stable CR3:C3d:CR2 complex suggests a molecular mechanism for the hand-over of CR3-bound immune complexes from macrophages to CR2-presenting cells in lymph nodes.Activation of complement leads to proteolytic cleavage of the central complement component, C3. Its major fragment, C3b, acts as an opsonin and becomes covalently bound to the activating surface via a reactive thioester located in the thioester (TE) domain of nascent C3b (Fig. S1A). Proteolytic processing by factor I within the CUB domain of C3b leads to the formation of iC3b and C3dg. Finally, C3d—which practically corresponds to the TE domain present in C3, C3b, and iC3b (Fig. S1 B–G)—is formed by other plasma proteases. These activation products are ligands for five complement receptors (1), with iC3b being the primary ligand of complement receptors (CRs) CR3 and CR4 (also known as CD11c/CD18, p150,95, or integrin α_Xβ₂), which is structurally similar to CR3.Like other integrins, CR3 is a heterodimeric complex of two transmembrane proteins, α_M and β₂. It is abundantly expressed on myeloid leukocytes, including neutrophil granulocytes, dendritic cells, monocytes, and macrophages and also on lymphoid natural killer (NK) cells (2). Most ligands, including iC3b (3), are bound by the Von Willebrand factor A (VWA) domain in the α-chain, also referred to as the αI domain owing to its insertion in the β-propeller domain. I domain residues coordinate a metal ion essential for ligand recognition through a metal ion-dependent adhesion site (MIDAS). Integrins adopt at least three major conformations in the cell membrane. The bent-closed conformation is inactive in ligand binding, the extended-closed conformation has low ligand affinity, and the extended-open conformation binds ligands with high affinity. The transition from the bent-closed to the open-extended conformation is exerted by a cytoplasmic force on the leg of the β-subunit, a process usually referred to as the inside-out signaling (4).Binding of ligands to CR3 leads to conformational changes in its ectodomain transmitting an outside-in signal through the cell membrane. This may lead to actin remodeling, phagocytosis, degranulation, and changes in leukocyte cytokine production (2, 5–7). CR3, and to a lesser degree CR4, are essential for the phagocytosis of complement-opsonized particles or complexes (6, 8, 9). Complement-opsonized immune complexes are captured in the lymph nodes by CR3-positive subcapsular sinus macrophages (SSMs) and conveyed directly to naïve B cells or through follicular dendritic cells (10) using CR1, CR2, and Fcγ receptors for antigen capture (11, 12). Hence, antigen-presenting cells such as SSMs may act as antigen storage and provide B lymphocytes with antigens (10, 12).Here, we establish the C3d fragment as the minimal and high-affinity binding partner for the CR3 I domain. By contrast, the binding site for the CR4 I domain was located in the C3c fragment by electron microscopy (13). We present the crystal structure of the CR3 I domain in complex with C3d. The classic observation of CR3 binding to iC3b, but not to its precursor C3b (14), is consistent with our structure. In addition, our structure and functional data suggest simultaneous binding of CR3 and another complement receptor, CR2, to C3 fragments, which might provide the basis for trafficking of complement-opsonized immune complexes from macrophages to B cells and follicular dendritic cells in lymph nodes.     

Sequence of the supernumerary B chromosome of maize provides insight into its drive mechanism and evolution

B chromosomes are enigmatic elements in thousands of plant and animal genomes that persist in populations despite being nonessential. They circumvent the laws of Mendelian inheritance but the molecular mechanisms underlying this behavior remain unknown. Here we present the sequence, annotation, and analysis of the maize B chromosome providing insight into its drive mechanism. The sequence assembly reveals detailed locations of the elements involved with the cis and trans functions of its drive mechanism, consisting of nondisjunction at the second pollen mitosis and preferential fertilization of the egg by the B-containing sperm. We identified 758 protein-coding genes in 125.9 Mb of B chromosome sequence, of which at least 88 are expressed. Our results demonstrate that transposable elements in the B chromosome are shared with the standard A chromosome set but multiple lines of evidence fail to detect a syntenic genic region in the A chromosomes, suggesting a distant origin. The current gene content is a result of continuous transfer from the A chromosomal complement over an extended evolutionary time with subsequent degradation but with selection for maintenance of this nonvital chromosome.

Supernumerary chromosomes were first discovered in the leaf-footed plant bug Metapodius more than a century ago (1). Since then, they have been reported in numerous plant, animal, and fungal species (2). A common feature of these so-called B chromosomes is that they are nonessential and are present only in some individuals in the population of a particular species. Through their evolution, they have developed various modes of behavior, e.g., tissue-specific elimination in Aegilops (3), preferential fertilization in Zea (4), or sex manipulation in Nasonia (5). In many plant species, they undergo controlled nondisjunction—unequal allocation to daughter nuclei during postmeiotic divisions (6). Their effect on frequency and distribution of meiotic crossovers along the standard A chromosomes has also been described (7, 8). Despite the peculiar behavior and unclear origins, no high-quality B chromosome reference sequence has been previously obtained in any organism.The B chromosome of maize is one of the most thoroughly studied supernumerary chromosomes (9–11) (Fig. 1). It can be found in numerous landraces and also in populations of Mexican teosinte, the maize wild relative (12). Despite being dispensable, it is maintained in populations by two properties: nondisjunction at the second pollen mitosis giving rise to unequal sperm and then preferential fertilization of the egg by the B chromosome-containing sperm (4, 13) (Fig. 1). This acrocentric chromosome is smaller than standard A chromosomes. Its long arm comprises proximal (PE) and distal euchromatin (DE), proximal heterochromatin (PH), and four large distal blocks of heterochromatin (DH1-4) (Fig. 1). Its short arm is minute. In a majority of genetic backgrounds, the presence of B chromosomes is not detrimental unless at high copy number (10), but in some others will cause breakage at the second pollen mitosis of some A chromosomes that contain heterochromatic knobs (14). This effect is thought to be an extension of the B chromosome drive mechanism involved with nondisjunction at this particular mitosis. The B chromosome has also evolved the property of increasing recombination in heterochromatic regions in general, probably to ensure its own chiasmata for proper distribution in meiosis (7, 15–17). Further, it has acquired a mechanism for its faithful transmission as a univalent (18, 19), which would also enhance its transmission, given that it can sometimes be present in odd numbers. Thus, classical cytogenetic studies established multiple properties of this unusual chromosome that act to maintain it in populations, but the molecular basis of its non-Mendelian inheritance remained obscure. To date, the efforts to generate B-specific sequences in maize have been limited to DNA marker development, general characterization of repetitive sequences, and low-pass sequencing (20–23). While genomes of several maize lines were sequenced to reference quality (24–26), comparable information for the supernumerary chromosome has not yet been available.Open in a separate windowFig. 1.The maize B chromosome. (A) Root tip metaphase spread of a line possessing nine B chromosomes (red signal). The red signal identifies the ZmBs B chromosome repeat in and around the centromere with a minor representative at the distal tip of the B long arm. Green signal identifies several chromosomal features, namely, the CentC centromeric satellite, 45S ribosomal DNA repeats, and TAG microsatellite clusters. DAPI stains the chromosomes (blue). (B) Schematic view of the acrocentric maize B chromosome at pachynema of meiosis. The chromosome is divided into the B short arm (BS), B centromere (BC), proximal heterochromatin (PH), proximal euchromatin (PE), four blocks of distal heterochromatin (DH1-4), and the distal euchromatin (DE). The B-specific repeat ZmBs, CentC satellite, CRM2 retrotransposon, knob heterochromatin, and TAG microsatellite cluster are color coded along the length of the chromosome. (C) Depiction of nondisjunction of the B chromosome. The B chromosome (blue with a red centromere) is shown in the generative nucleus (G) after the first microspore division. After replication, the two chromatids proceed to the same pole at the second microspore mitosis in the vast majority of divisions. Thus, most mature pollen grains contain two sperm (S) with only one containing the B chromosomes. V: vegetative cell. (D) Depiction of preferential fertilization. For most lines of maize, the sperm with the two B chromosomes will preferentially fertilize the egg (E) as compared with the central cell (C) in the process of double fertilization. The fertilized egg develops into the next generation embryo and the fertilized central cell develops into the endosperm. The combination of nondisjunction at the second pollen mitosis and preferential fertilization comprise the drive mechanism of the B chromosome.     

Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components

The enzymatic degradation of recalcitrant plant biomass is one of the key industrial challenges of the 21st century. Accordingly, there is a continuing drive to discover new routes to promote polysaccharide degradation. Perhaps the most promising approach involves the application of "cellulase-enhancing factors," such as those from the glycoside hydrolase (CAZy) GH61 family. Here we show that GH61 enzymes are a unique family of copper-dependent oxidases. We demonstrate that copper is needed for GH61 maximal activity and that the formation of cellodextrin and oxidized cellodextrin products by GH61 is enhanced in the presence of small molecule redox-active cofactors such as ascorbate and gallate. By using electron paramagnetic resonance spectroscopy and single-crystal X-ray diffraction, the active site of GH61 is revealed to contain a type II copper and, uniquely, a methylated histidine in the copper's coordination sphere, thus providing an innovative paradigm in bioinorganic enzymatic catalysis.     

Cargo recognition mechanism of myosin X revealed by the structure of its tail MyTH4-FERM tandem in complex with the DCC P3 domain

Myosin X (MyoX), encoded by Myo10, is a representative member of the MyTH4-FERM domain-containing myosins, and this family of unconventional myosins shares common functions in promoting formation of filopodia/stereocilia structures in many cell types with unknown mechanisms. Here, we present the structure of the MyoX MyTH4-FERM tandem in complex with the cytoplasmic tail P3 domain of the netrin receptor DCC. The structure, together with biochemical studies, reveals that the MyoX MyTH4 and FERM domains interact with each other, forming a structural and functional supramodule. Instead of forming an extended β-strand structure in other FERM binding targets, DCC_P3 forms a single α-helix and binds to the αβ-groove formed by β5 and α1 of the MyoX FERM F3 lobe. Structure-based amino acid sequence analysis reveals that the key polar residues forming the inter-MyTH4/FERM interface are absolutely conserved in all MyTH4-FERM tandem-containing proteins, suggesting that the supramodular nature of the MyTH4-FERM tandem is likely a general property for all MyTH4-FERM proteins.