Nascent Septa Research Articles

Spatial and temporal localization of cell wall associated pili in Enterococcus faecalis.

Enterococcus faecalis virulence requires cell wall-associated proteins, including the sortase-assembled endocarditis and biofilm associated pilus (Ebp), important for biofilm formation in vitro and in vivo. The current paradigm for sortase-assembled pilus biogenesis in Gram-positive bacteria is that sortases attach substrates to lipid II peptidoglycan (PG) precursors, prior to their incorporation into the growing cell wall. Contrary to prevailing dogma, by following the distribution of Ebp and PG throughout the E. faecalis cell cycle, we found that cell surface Ebp do not co-localize with newly synthesized PG. Instead, surface-exposed Ebp are localized to the older cell hemisphere and excluded from sites of new PG synthesis at the septum. Moreover, Ebp deposition on the younger hemisphere of the E. faecalis diplococcus appear as foci adjacent to the nascent septum. We propose a new model whereby sortase substrate deposition can occur on older PG rather than at sites of new cell wall synthesis. Consistent with this model, we demonstrate that sequestering lipid II to block PG synthesis via ramoplanin, does not impact new Ebp deposition at the cell surface. These data support an alternative paradigm for sortase substrate deposition in E. faecalis, in which Ebp are anchored directly onto uncrosslinked cell wall, independent of new PG synthesis.

Localization of the key cell division determinant FtsZ in Francisella tularensis.

Francisella tularensis, the causative agent of tularemia, is a highly infectious gram-negative bacterium capable of replicating within macrophages, leukocytes, dendritic cells and epithelial cells. In addition, F. tularensis also invades erythrocytes. FtsZ, a homologue of eukaryotic tubulin, is an essential cell division protein in almost all bacteria. Polymerization of FtsZ into a contractile ring is a critical first step in division and the FtsZ ring acts in recruiting the cell wall synthesis machinery to the nascent septum. We have generated a recombinant fusion of ftsZftto emgfp (Emerald Green Fluorescent Protein) which can be expressed, in trans, under its presumed native promotor or a strong Francisella promoter (pGRP). As expected, FtsZft-Emgfp can be seen localizing as rings in F. tularensis. Our aim is to utilize fluorescently tagged FtsZ to investigate replication of F. tularensis in a range of in vitro host cell invasion assays and as a marker for viability to study the morphological and physiological differentiation of F. tularensis as it transitions into a viable but non-culturable (VBNC) state. (This research was made possible by NASA West Virginia Space Grant Consortium Training Grant #NNX15A101H and by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence).

Coordination of Growth, Chromosome Replication/Segregation, and Cell Division in E. coli.

Bacterial cells growing in steady state maintain a 1:1:1 relationship between an appropriate mass increase, a round of DNA replication plus sister chromosome segregation, and cell division. This is accomplished without the cell cycle engine found in eukaryotic cells. We propose here a formal logic, and an accompanying mechanism, for how such coordination could be provided in E. coli. Completion of chromosomal and divisome-related events would lead, interactively, to a “progression control complex” (PCC) which provides integrated physical coupling between sister terminus regions and the nascent septum. When a cell has both (i) achieved a sufficient mass increase, and (ii) the PCC has developed, a conformational change in the PCC occurs. This change results in “progression permission,” which triggers both onset of cell division and release of terminus regions. Release of the terminus region, in turn, directly enables a next round of replication initiation via physical changes transmitted through the nucleoid. Division and initiation are then implemented, each at its own rate and timing, according to conditions present. Importantly: (i) the limiting step for progression permission may be either completion of the growth requirement or the chromosome/divisome processes required for assembly of the PCC; and, (ii) the outcome of the proposed process is granting of permission to progress, not determination of the absolute or relative timings of downstream events. This basic logic, and the accompanying mechanism, can explain coordination of events in both slow and fast growth conditions; can accommodate diverse variations and perturbations of cellular events; and is compatible with existing mathematical descriptions of the E. coli cell cycle. Also, while our proposition is specifically designed to provide 1:1:1 coordination among basic events on a “per-cell cycle” basis, it is a small step to further envision permission progression is also the target of basic growth rate control. In such a case, the rate of mass accumulation (or its equivalent) would determine the length of the interval between successive permission events and, thus, successive cell divisions and successive replication initiations.

Visualization of yeast cells by electron microscopy

In the 1970s, hydrocarbon or methanol utilizable yeasts were considered as a material for foods and ethanol production. During the course of studies into the physiology of yeasts, we found that these systems provide a suitable model for the biogenesis and ultrastructure research of microbodies (peroxisomes). Microbodies of hydrocarbon utilizing Candida tropicalis multiply profusely from the preexisting microbody. β oxidation enzymes in the microbody were determined by means of immunoelectron microscopy. We examined the ultrastructure of Candida boidinii microbodies grown on methanol, and found a composite crystalloid of two enzymes, alcohol oxidase and catalase, by analyzing using the optical diffraction and filtering technique and computer simulation. We established methods for preparing the protoplasts of Schizosaccharomyces pombe and conditions for the complete regeneration of the cell wall. The dynamic process of cell wall formation was clarified through our study of the protoplasts, using an improved ultra high resolution (UHR) FESEM S-900 and an S-900LV. It was found that β-1,3-glucan, β-1,6-glucan and α-1,3-glucan, as well as α-galactomannan, are ingredients of the cell wall. The process of septum formation during cell division was examined after cryo-fixation by high pressure freezing (HPF). It was also found that α-1,3- and β-1,3-glucans were located in the invaginating nascent septum, and later, highly branched β-1,6-glucan also appeared on the second septum. The micro-sampling method, using a focused ion beam (FIB), has been applied to our yeast cell wall research. A combination of FIB and scanning transmission electron microscopy is useful in constructing 3D images and analyzing the molecular architecture of cells, as well as for electron tomography of thick sections of biological specimens.

Negative membrane curvature as a cue for subcellular localization of a bacterial protein

Bacterial proteins often localize to distinct sites within the cell, but the primary cues that dictate localization are largely unknown. Recent evidence has shown that positive membrane curvature can serve as a cue for localization of a peripheral membrane protein. Here we report that localization of the peripheral membrane protein DivIVA is determined in whole or in part by recognition of negative membrane curvature and that regions of the protein near the N and C terminus are important for localization. DivIVA, which is a cell division protein in Bacillus subtilis, localizes principally as a ring at nascent septa and secondarily to the less negatively curved, inside surface of the hemispherical poles of the cell. When cytokinesis is prevented, DivIVA redistributes itself to, and becomes markedly enriched at, the poles. When the rod-shaped cells are converted into spheres (protoplasts) by treatment with lysozyme, DivIVA adopts a largely uniform distribution around the cell. Recognition of membrane curvature by peripheral membrane proteins could be a general strategy for protein localization in bacteria.

A class Vb chitin synthase in Colletotrichum graminicola is localized in the growing tips of multiple cell types, in nascent septa, and during septum conversion to an end wall after hyphal breakage

Previous complementation of a chitin synthase class Vb null mutant (Colletotrichum graminicola chsA) indicated that the encoded protein is responsible for approximately 30% of the conidial chitin, is essential for conidial wall strength in media with high water potential, and contributes to strength of hyphal tips. We complemented a chsA null mutant with chsA fused to the green-fluorescent protein (sgfp) gene driven by a heterologous constitutively expressed promoter. Comparisons of the strain with the ectopic chsA-sgfp to the wild type indicated that ChsA-sGFP serves the same biological functions as ChsA in that like the wild type, the chsADelta chsA::sgfp (EC) had conidia that did not explode and hyphal tips that did not swell. Confocal microscopy of ChsA-sGFP (EC) cells stained with the membrane stain FM 4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide) indicated that ChsA is localized in the plasma membrane of the following: growing apices of hyphal branches, conidiophores, and falcate and oval conidia; in nascent septa; and in septa that are being converted to an end wall after hyphal breakage. The data support the hypothesis that chsA either directly or indirectly encodes the information for its localization, that ChsA is localized in the plasma membrane, and that the class Vb enzyme produces chitin synthase in multiple cells and after wall breakage.

Septal localization of the SpoIIIE chromosome partitioning protein in Bacillus subtilis

The 787 amino acid SpoIIIE protein of Bacillus subtilis is required for chromosome partitioning during sporulation. This process differs from vegetative chromosome partitioning in that it occurs after formation of the septum, apparently by transfer of the chromosome through the nascent septum in a manner reminiscent of plasmid conjugation. Here we show that SpoIIIE is associated with the cell membrane, with its soluble C-terminal domain located inside the cell. Immunofluorescence microscopy using affinity-purified anti-SpoIIIE antibodies shows that SpoIIIE is targeted near the centre of the asymmetric septum, in support of a direct role for SpoIIIE in transport of DNA through the septum. We also report on the isolation of a mutation affecting the N-terminal hydrophobic domain of SpoIIIE that interferes with targeting to the septum and blocks DNA transfer. This mutation also causes de-localization of the activity of the normally prespore-specific sigma factor, sigmaF, consistent with the notion that SpoIIIE can form a seal between the chromosomal DNA and the leading edge of the division septum.

Role of murein lipoprotein in morphogenesis of the bacterial division septum: phenotypic similarity of lkyD and lpo mutants

Phenotypes were compared in two different classes of mutants with defects in murein-lipoprotein (lkyD mutants of Salmonella typhimurium and an lpo mutant of Escherichia coli). Both mutations are associated with the same triad of phenotypic abnormalities, consisting of defective formation of the division septum, leakage of periplasmic proteins during growth, and increased sensitivity to several unrelated external toxic agents. The abnormality in septum formation consists of a defect in invagination of the outer membrane during formation of the nascent septum. The results suggest that formation of the murein-lipoprotein link plays an important role in differentiation of the division septum and perhaps also in maintaining the normal barrier function of the outer membrane.

Morphogenesis of the bacterial division septum: a new class of septation-defective mutants.

A new class of mutants of Salmonella typhimurium (lkyD mutants) are described. The mutants are defective in morphogenesis of the division septum, and are characterized by a failure of the outer membrane to invaginate despite normal ingrowth of the cytoplasmic membrane and murein layers of the growing septum. The cell envelopes of the mutants show a significant decrease in the bound form of murein-lipoprotein and a corresponding increase in the free form of the lipoprotein. This suggests that the morphogenic defect may result from a defect in formation of covalent bonds between the free lipoprotein of the outer membrane and the murein of the nascent septum.

Effect of Inhibition of Deoxyribonucleic Acid and Protein Synthesis on the Direction of Cell Wall Growth in Streptococcus faecalis

Selective inhibition of protein synthesis in Streptococcus faecalis (ATCC 9790) was accompanied by a rapid and severe inhibition of cell division and a reduction of enlargement of cellular surface area. Continued synthesis of cell wall polymers resulted in rapid thickening of the wall to an extent not seen in exponential-phase populations. Thus, the normal direction of wall growth was changed from a preferential feeding out of new wall surface to that of thickening existing cell surfaces. However, the overall manner in which the wall thickened, from nascent septa toward polar regions, was the same in both exponential-phase and inhibited populations. In contrast, selective inhibition of deoxyribonucleic acid (DNA) synthesis using mitomycin C was accompanied by an increase in cellular surface area and by division of about 80% of the cells in random populations. Little or no wall thickening was observed until the synthesis of macromolecules other than DNA was impaired and further cell division ceased. Concomitant inhibition of both DNA and protein synthesis inhibited cell division but permitted an increase in average cell volume. In such doubly inhibited cells, walls thickened less than in cells inhibited for protein synthesis only. On the basis of the results obtained, a model for cell surface enlargement and cell division is presented. The model proposes that: (i) each wall enlargement site is influenced by an individual chromosome replication cycle; (ii) during chromosome replication peripheral surface enlargement would be favored over thickening (or septation); (iii) a signal associated with chromosome termination would favor thickening (and septation) at the expense of surface enlargement; and (iv) a factor or signal related to protein synthesis would be required for one or more of the near terminal stages of cell division or cell separation, or both.