Strength of concave septa and depth limits of fossil cephalopods

Westermann, G. E. G.: Strength of concave septa and depth limits of fossil cephalopods.
Simple septa with spherical curvature are present in the shells of all Endocer-oidea, Actinoceroidea, Bactritoidea, and most Nautiloidea and Coleoidea. Such septa act as quasi-hemispherical concave membranes when subjected to hydrostatic pressure. Since the tensile strength of a spherical membrane is directly proportional to the ratio of its thickness and radius of curvature, measurements of these parameters on polished and thin sections of septa can be used to obtain strength of the septum against implosion. Depth limits of fossil cephalopods can be made by calibrating these measurements in terms of recent implosion data on 'living' Spirula and Nautilus . Estimates of septal strength are augmented by strength estimates for long septal necks and cylindrical to globular connecting rings.
Assuming that actual habitats ranged to approximately two-thirds of the mechanical limits of the shells, the following maximum depth ranges are indicated from this preliminary survey: Endoceroidea 100–450 m; Actinoceroidea 50–150 m; Nautiloidea, Ellesmerocerida 50–200 m, Orthocerida 150–500 m, Oncocer-ida <150, Discosorida <100 m, Tarphycerida <150 m, Nautilida 200–600 m; Bactritoidea c. 400 m; Coleoidea, Aulacocerida 200–900 m, Sepiida 200–1000 m, Belemnitida 50–200 exceptionally 350 m.     

Post-mortem descent with septal implosion in Silurian nautiloids

Late Silurian nautiloids from Bohemia have either (1) thin and densely spaced septa of which many are broken and internally accumulated, or (2) thick and widely spaced septa wnich are all intact. Since the latest chamber (s) and the shell wall are undamaged, septal fragmentation occurred by implosion during postmortem sinking, with sea water rushing in through the siphuncle. The latest connecting ring(s) were more permeable than the immature ones, permitting pressure compensation in the latest chamber(s). Septal debris accumulated adapically indicating the (ultimate) sinking orientation. Depth (maxima) of the nautiloid habitats and of the Bohemian basin are estimated from the strength parameters of the broken and intact septa: brevicones — epipelagic, weak longicones — moderately shallow pelagic, strong longicones — nektobenthic, sea floor depth — several 100 m. Silurian-Nautiloids-Shell-Connecting rings-Bathymetry.     

Distribution of elastin and collagen in canine lung alveolar parenchyma

We have quantified the fibrous collagen (predominantly type I) and elastin in four locations of perceived mechanical importance: one quasi-planar feature, the alveolar septum or wall (W), and three linear features, the junction (J) of three septa, the free edges (E) of septa, and the line along which two septa join at a distinct angle or bend (B). The frequencies of these four features on light micrographs and the areas of transections through collagen and elastin seen on electron micrographs were combined to give the volumes of collagen and elastin within each feature. We find that E and B have similar compositions and contain most (4/5) of the parenchymal elastin in their relatively heavy cables. The E and B are interconnected and similar in location and composition, and they may constitute a functional entity in which elastin provides tension over a range of lung volumes, opposing septal tensions. In J and W, elastin is typically sparse and fine. Calculations, however, suggest it contributes the dominant portion of septal tension at lower lung volumes. Elastin may be essential to stabilizing septal configuration. Collagen, on the other hand, is distributed relatively evenly throughout E, B, J, and W, consistent with the role of protecting all components against rupture.     

Does elevated wall tension cause aortic aneurysm rupture? Investigation using a subject-specific heterogeneous model

ObjectiveTo investigate whether peak wall tension in abdominal aortic aneurysm occurs at the site of rupture to test for a causative relationship.MethodsFour ruptured and nine unruptured AAA were harvested whole from cadavers, followed by regional measurements of wall thickness, elastic parameters and failure tension. Finite element models were developed with subject-specific load-free AAA morphology and heterogeneous properties interpolated using a geodesic distance weighted approach from the measurements. The wall tension under uniform pressure and tension to failure tension ratio as an index of susceptibility to rupture were computed. As a secondary aim, the peak wall tension using this heterogeneous model approach was compared to the traditional homogeneous model approach in order to evaluate the reliability of the latter.ResultsThe average peak wall tension in the ruptured group was 43% higher than in the unruptured group without statistical significance even though it was 54% larger in diameter. The site of peak wall tension was in the vicinity of the site of rupture in two ruptured AAA. The peak tension did not breach failure tension at the rupture site in any of the AAA. The traditional population-wide homogeneous model approach overestimated peak wall tension by just 3% compared to the subject-specific heterogeneous model approach.ConclusionWe failed to find adequate evidence of a causative relationship between peak wall tension and AAA rupture. The findings are not conclusive owing to study limitations such as ignoring intraluminal thrombus, sparse distribution of specimens procured and small study population.     

Septum-directed secretion in the filamentous fungus Aspergillus oryzae

Although exocytosis in fungal cells takes place at hyphal tips, there also seems a line of circumstantial evidence suggesting the occurrence of exocytosis at other sites of cells, such as septa. To investigate whether exocytosis takes place at fungal septa, we monitored dynamics of EGFP‐fused α‐amylase (AmyB–EGFP), the representative secretory enzyme of the filamentous fungus Aspergillus oryzae. We found that AmyB–EGFP accumulates in Spitzenkörper at hyphal tips as well as septal periplasm between the plasma membrane and cell walls. The septal accumulation of AmyB–EGFP was a rapid process, and required microtubules but not F‐actin. Thus, this process is independent of exocytosis at hyphal tips that requires both microtubules and F‐actin. In addition, fluorescence recovery after photobleaching (FRAP) analysis of EGFP‐fused AoSnc1 revealed that secretory vesicles constitutively fuse with the septal plasma membrane. These results demonstrated that exocytosis takes place at septa in addition to hyphal tips. Analysis of two plasma membrane transporters, AoUapC and AoGap1, revealed that they preferentially accumulate at septa and the lateral plasma membrane with no clear accumulation at apical Spitzenkörper, suggesting that non‐tip directed exocytosis is important for delivery of these proteins.     

Pentapeptide-repeat,cytoplasmic-membrane protein HglK influences the septal junctions in the heterocystous cyanobacterium Anabaena

N₂-fixing heterocystous cyanobacteria grow as chains of cells that are connected by proteinaceous septal junctions, which traverse the septal peptidoglycan through nanopores and mediate intercellular molecular transfer. In the model organism Anabaena sp. strain PCC 7120, proteins SepJ, FraC and FraD, which are localized at the cell poles in the intercellular septa, are needed to produce septal junctions. The pentapeptide-repeat, membrane-spanning protein HglK has been described to be involved in the deposition of the heterocyst-specific glycolipid layer, but the hglK mutant also showed intercellular septa broader than in the wild type. Here we found that hglK mutant of Anabaena is impaired in the expression of heterocyst-related genes coxB2A2C2 (cytochrome c oxidase) and nifHDK (nitrogenase), indicating a defect in heterocyst differentiation. HglK was predominantly localized at the intercellular septa and was required to make long filaments, produce a normal number of nanopores and express full intercellular molecular transfer activity. However, the effects of hglK inactivation were not additive to those of the inactivation of sepJ and/or fraC-fraD. We suggest that HglK contributes to the architecture of the intercellular septa with an impact on the function of septal junctions.     

Temporal expression of alpha-smooth muscle actin and drebrin in septal interstitial cells during alveolar maturation.

In rat lung, the definitive alveoli are established during development by the outgrowth of secondary septa from the primary septa present in newborn; however, the mechanism of alveolar formation has not yet been fully clarified. In this study, we characterize the septal interstitial cells in developing alveoli. During the perinatal period, alpha-SMA-containing slender cells were found in the primitive alveolar septa. Alpha-SMA-containing cells were detected at the tips of the septa until postnatal day 21, when the alveolar formation was almost completed, but disappeared in adult. Immunoelectron microscopy demonstrated that alpha-SMA is localized mainly in the cellular protrusions, which are connected with the elastic fibers around the interstitial cells. Developmentally regulated brain protein (drebrin) is also located in the cell extensions containing alpha-SMA in immature alveolar interstitial cells. In adult lung, alpha-SMA-positive cells are located only at the alveolar ducts but are not found in the secondary septa. Desmin is expressed only in alpha-SMA-containing cells at the alveolar ducts but not in those at the tip of alveolar septa. These results suggest that a part of the septal interstitial cells are temporarily alpha-SMA- and drebrin-positive during maturation. Alpha-SMA- and drebrin-containing septal interstitial cells (termed septal myofibroblast-like cells) may play an important role in alveolar formation.     

Acellular hemoglobin solution enters compressed lung capillaries more readily than red blood cells.

High lung inflation pressures compress alveolar septal capillaries, impede red cell transit, and interfere with oxygenation. However, recently introduced acellular hemoglobin solutions may enter compressed lung capillaries more easily than red blood cells. To test this hypothesis, we perfused isolated rat lungs with fluorescently labeled diaspirin cross-linked hemoglobin (DCLHb; 10%) and/ or autologous red cells (hematocrit, 20). Septal capillaries were compressed by setting lung inflation pressure above vascular pressures (zone 1). Examination by confocal microscopy showed that DCLHb was distributed throughout alveolar septa. Furthermore, this distribution was not affected by adding red blood cells to the perfusate. We estimated the maximum acellular hemoglobin mass within septa to be equivalent to that of 15 red blood cells. By comparison, we found an average of 2.7 +/- 4.6 red cells per septum in zone 1. These values increased to 30.4 +/- 25.8 and 50.4 +/- 22.1 cells per septum in zones 2 and 3, respectively. We conclude that perfusion in zone 1 with a 10% acellular hemoglobin solution may increase the hemoglobin concentration per septum up to fivefold compared with red cell perfusion.     

Ontogeny of 'epithecal' and septal structures in scleractinian corals

The ontogenetic development of a solitary scleractinian coral, Flabellum distinctum Edwards & Haime, has been studied in serial thin section, with special attention being paid to epithecal nature in relation to septal growth. The term 'epitheca' has been confusingly used for two different skeletal structures: epitheca ( sensu stricto ) and marginotheca. The latter is here newly proposed. 'epitheca' is defined as a calcareous investment developed on the outside of other skeletal structures of a corallite. It can be distinguished from the marginotheca in section by lacking a dark line (calcification centre) and by being unrelated to the formation of septa. 'marginotheca' defines the outer margin of the main skeletal structures of a corallite. It has a dark line which functionally coincides with that of the eutheca. It is of primary origin, preceding formation of septa and provides the origin of the septa. The marginotheca is one of the more important and fundamental skeletal structures for coral classification.     

Origin and phylogeny of Guyniidae (Scleractinia) in the light of microstructural data

The set of skeletal characters of the Recent azooxanthellate coral Guynia annulata Duncan, 1872 is unique among extant scleractinians and encompasses: (a) undifferentiated septal calcification centers (in most extant scleractinians calcification centers are clearly separated); (b) completely smooth septal faces (septa of almost all extant scleractinians bear granular ornamentation); (c) deeply recessed septa in respect to the epithecal rim in the adult coralla (in adults of the majority of extant scleractinians the relationships between septa and wall are the reverse); and (d) an aseptal part of the initial ontogenetic stage, just above the basal plate (almost all known scleractinians have a septate initial coralla). Skeletal features of five other extant traditional guyniids are typical of other caryophylliines (and of Scleractinia). However, the wall types present in different species of traditional guyniids exceed limits traditionally attributed to one caryophylliine family: i.e., Stenocyathus and Truncatoguynia have a marginothecal wall like the Flabellidae, whereas Schizocyathus and Temnotrochus usually have an entirely epithecal wall, as in Gardineriidae (Volzeioidea). Moreover, Pourtalocyathus and Schizocyathus show intraspecific variation in distribution of septal calcification centers (separated vs. non-separated) and in wall types (epithecal vs. consisting of large spherulite-like bodies). These major differences in skeletal architecture form the basis for a new, threefold taxonomical subdivision of the traditional guyniids: (1) Guyniidae Hickson, 1910, containing only monospecific Guynia with an epithecal wall, and septa with non-separated calcification centers; (2) Schizocyathidae fam.n., groups Microsmilia Schizocyathus, Pourtalocyathus, Temnotrochus, which have an epithecal wall and septa with usually well-separated calcification centers; and (3) Stenocyathidae fam.n. with Stenocyathus and Truncatoguynia which have a marginothecal wall and septa with well-separated calcification centers. Despite differences in the basic architecture of the skeleton, all taxa attributed to these families have 'thecal pores' formed by selective dissolution of the skeleton. I propose two hypotheses for evolutionary relationships among Guyniidae, Schizocyathidae, and Stenocyathidae: (1) Hypothesis A: the three families are not phylogenetically related and 'pores' originated independently in different scleractinian lineages: e.g., Guyniidae may represent distant zardinophyllid or gigantostyliid descendants, Schizocyathidae may be a volzeioid offshoot, whereas Stenocyathidae may be a flabellid descendant; (2) Hypothesis B: the three families are phylogenetically related and 'thecal pores' are synapomorphic for the clade (superfamily Guynioidea). Additional approaches, such as anatomical observations, molecular studies on guyniid DNA sequences, and in-depth studies on scleractinian biomineralization will be necessary to test these hypotheses.     

A Highlights from MBoC Selection: Mitotic regulation of fungal cell-to-cell connectivity through septal pores involves the NIMA kinase

Intercellular bridges are a conserved feature of multicellular organisms. In multicellular fungi, cells are connected directly via intercellular bridges called septal pores. Using Aspergillus nidulans, we demonstrate for the first time that septal pores are regulated to be opened during interphase but closed during mitosis. Septal pore–associated proteins display dynamic cell cycle–regulated locations at mature septa. Of importance, the mitotic NIMA kinase locates to forming septa and surprisingly then remains at septa throughout interphase. However, during mitosis, when NIMA transiently locates to nuclei to promote mitosis, its levels at septa drop. A model is proposed in which NIMA helps keep septal pores open during interphase and then closed when it is removed from them during mitosis. In support of this hypothesis, NIMA inactivation is shown to promote interphase septal pore closing. Because NIMA triggers nuclear pore complex opening during mitosis, our findings suggest that common cell cycle regulatory mechanisms might control septal pores and nuclear pores such that they are opened and closed out of phase to each other during cell cycle progression. The study provides insights into how and why cytoplasmically connected Aspergillus cells maintain mitotic autonomy.     

Lengths and topology of alveolar septal borders

To clarify the mechanics of alveolar parenchyma, we undertook a stereological and topological study in perfusion-fixed canine lungs of the borders of alveolar septa. We defined the principal borders as those along which one septum 1) joins two others (J), 2) joins one other at a distinct angle (B), or 3) joins no other structure (E). E and B borders are invariably reinforced with heavy connective tissue cables; J borders are not. Relative net lengths, determined from the number of traces per section area, were J, 45%; E, 19%; and B, 25%. These were remarkably constant over 10 canine lobes (5 animals, 4 volumes). Parenchyma, then, departs from the simple models that comprise only Js and Es. Bs are important; their net length exceeds that of Es. With lobe deflation, E shortened somewhat more than required to maintain geometric similarity, suggesting that the alveolar duct contracted disproportionately. A three-dimensional reconstruction was made from serial sections, and individual border segments were followed through the reconstruction. Typical lengths of individual J, B, and E borders were nearly equal. To characterize how the network of borders were interconnected, we counted the nodes at which they meet by class, e.g., EBE for the meeting of one B, two Es. The most common are JJJJ, 26%; EEEJ, 10%; EBJ, 24%; EBE, 8%; BBJJ, 12%. If parenchyma were constructed only from free-standing entrance rings and septal junctions, only JJJJ and EEEJ would be anticipated. The presence of EBJ, EBE, and BBJJ underscores parenchymal complexity. Only 7% of septa examined were bordered entirely by Js. Connective tissue cables were not confined to the alveolar duct's lumen but often extended to the primary septa at the periphery of the ductal unit. They rarely linked adjacent alveolar ducts; only 1 in 200 cable segments crossed from one duct to another. These observations support the concept that the parenchyma is an elastic network, characterized in part by a serial mechanical linkage from connective tissue cable to septal membrane to cable again.     

Septal implosion in Late Carboniferous coiled nautiloids from Ohio

Mapes, R.H. & McComas, G.A. 2010: Septal implosion in Late Carboniferous coiled nautiloids from Ohio. Lethaia, 10.1111/j.1502–3931.2009.00213.x More than 200 relatively mature coiled nautiloid specimens, assigned to Metacoceras mcchesneyi, were recovered from an Upper Carboniferous shale in northeastern Ohio. Twenty‐seven undistorted specimens reveal that the septa in every specimen were collapsed and/or telescoped. This septal collapse without external shell distortion could only have been accomplished by limited implosion due to excessive pressure. Analysis of the fossils, sediment and the depositional environment indicate that after burial, the nautiloid cameral spaces were probably filled with both liquid and gas, and the body chamber was filled with semi‐solid thixotropic mud. To prevent conch collapse at the time of septal implosion, the thixotropic mud filling the nautiloid body chamber acted as a liquid at the time of stress release during septal failure. The stress was produced by combined lithostatic and hydrostatic pressures, which fluidized the unlithified thixotropic mud that flowed from the body chamber into the phragmocone during septal collapse. After the septal implosion and when flowage ceased, the thixotropic mud quickly resolidified into a solid state providing internal conch support that prevented the collapse of the conch. □Carboniferous, nautiloids, septal implosion, taphonomy, thixotropic mud.     

Skeletal ontogeny in basal scleractinian micrabaciid corals

The skeletal ontogeny of the Micrabaciidae, one of two modern basal scleractinian lineages, is herein reconstructed based on serial micro‐computed tomography sections and scanning electron micrographs. Similar to other scleractinians, skeletal growth of micrabaciids starts from the simultaneous formation of six primary septa. New septa of consecutive cycles arise between septa of the preceding cycles from unique wedge‐shaped invaginations of the wall. The invagination of wall and formation of septa are accompanied by development of costae alternating in position with septa. During corallite growth, deepening invagination of the wall results in elevation of septa above the level of a horizontal base. The corallite wall is regularly perforated thus invaginated regions consist of pillars inclined downwardly and outwardly from the lower septal margins. Shortly after formation of septa (S2 and higher cycles) their upper margins bend and fuse with the neighboring members of a previous cycle, resulting in a unique septal pattern, formerly misinterpreted as “septal bifurcation.” Septa as in other Scleractinia are hexamerally arranged in cycles. However, starting from the quaternaries, septa within single cycles do not appear simultaneously but are inserted in pairs and successively flank the members of a preceding cycle, invariably starting from those in the outermost parts of the septal system. In each pair, the septum adjacent to older septa arises first (e.g., the quinaries between S2 and S4 before quinaries between S3 and S4). Unique features of micrabaciid skeletal ontogeny are congruent with their basal position in scleractinian phylogeny, which was previously supported by microstructural and molecular data. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

Internal shell destruction in some Silurian nautiloids

Internal destruction of septa in Silurian brevicone cephalopods has taken place during post-mortem descent. The breakage was caused by implosions due to increasing water pressure. A pre-vious interpretation involving instantaneous internal collapse of septa is discussed and opposed. Due to shell construction, the surviving parts of broken septa, and broken septal fragments contained within the shell, the breakage of septa must have taken place in one chamber at a time, with a speed controlled by the water seeping in through the partly blocked siphuncle. The destruction could occasionally leave isolated septa unbroken, which confirms an interpretation of repeated breakage. As the destruction of septa has taken some time it can not be excluded that shells have undergone post-mortem drift.     

A contribution to knowledge of the compartments and the fascial and septal formations of the popliteal fossa in the human fetus and the adult.

A contribution to knowledge of the compartments and the fascial and septal formations of the popliteal fossa in the human fetus and the adult. A study was made in human fetuses from the 3rd month onwards, newborn and the adult of the fascial and septal formations and the compartments of the popliteal fossa. Observations of serial sections of the knee of human fetuses, of macroscopic preparations of the knee of newborns and of ultrasound images of the popliteal fossa in adults showed that: the fascial formation covering the popliteal fossa consists of the popliteal fascia and the superficial fascia. The bud of the popliteal fascia is observable in the 3-month fetus as a layer of thin fibrillar connective tissue which is thicker in the tracts between the muscle buds. At birth the popliteal fascia is clearly a separate anatomical entity of continuous laminar structure which is thicker in the tracts between the muscles and thinner where it covers them. The superficial fascia becomes evident in fetuses at a later stage (6th month) in the form of a thin lamina in the frontal plane which at birth is well defined and observable as a thin continuous line deep below the subcutaneous layer. The septal formation consists of four septa: two in the sagittal plane (lateral and medial) and two in the frontal plane (lateral and medial). The bud of these septa appears in 4-month fetuses after the appearance of the popliteal fascia. They branch off from the thicker connective areas between the muscles buds as connective prolongations which later assume a laminar aspect and eventually become compact and form septa. In at-term fetuses and newborns these septal formations are clearly recognizable as antomical entities, which branch off from the deep surface of the thicker tracts of the popliteal fascia and are inserted into the femur. The relationships and connections with the muscular groups are also clearly visible. The organization and demarcation of the compartments, which is already delineated in the 6-month fetus, seems to be completed at birth, considering the presence of the superficial fascia, the popliteal fascia and the septa. It is possible to distinguish a superficial compartment between the popliteal and the superficial fascia an a deep compartment between the frontal septa, the skeletal plane and the popliteal fascia. This deep compartment is clearly subdivided by the two sagittal septa into three sectors (medial, intermediate and lateral). The medial and lateral sectors contain muscles, while the intermediate compartment contains the vasculonervous bundle and the popliteal adipose body.     

Nucleoid-independent identification of cell division sites in Escherichia coli

The mechanism used by Escherichia coli to determine the correct site for cell division is unknown. In this report, we have attempted to distinguish between a model in which septal position is determined by the position of the nucleoids and a model in which septal position is predetermined by a mechanism that does not involve nucleoid position. To do this, filaments with extended nucleoid-free regions adjacent to the cell poles were produced by simultaneous inactivation of cell division and DNA replication. The positions of septa that formed within the nucleoid-free zones after division was allowed to resume were then analyzed. The results showed that septa were formed at a uniform distance from cell poles when division was restored, with no relation to the distance from the nearest nucleoid. In some cells, septa were formed directly over nucleoids. These results are inconsistent with models that invoke nucleoid positioning as the mechanism for determining the site of division site formation.     

Rupture of the cell envelope by induced intracellular gas phase expansion in gas vacuolate bacteria.

Using a new approach, we estimated the physical strength of the cell envelopes of three species of gram-negative, gas vacuolate bacteria (Microcyclus aquaticus, Prosthecomicrobium pneumaticum, and Meniscus glaucopis). Populations of cells were slowly (0.5 to 2.9 h) saturated with argon, nitrogen, or helium to final pressures up to 100 atm (10, 132 kPa). The gas phases of the vesicles remained intact and, upon rapid (1 to 2 s) decompression to atmospheric pressure, expanded and ruptured the cells; loss of colony-forming units was used as an index of rupture. Because the cell envelope is the cellular component most likely to resist the expanding intracellular gas phase, its strength can be estimated from the minimum gas pressures that produce rupture. The viable counts indicated that these minimum pressures were between 25 and 50 atm; the majority of the cell envelopes were ruptured at pressures between 50 and 100 atm. Cells in which the gas vesicles were collapsed and the gas phases were effectively dissolved by rapid compression tolerated decompression from much higher gas saturations. Cells that do not normally possess gas vesicles (Escherichia coli) or that had been prevented from forming them by addition of L-lysine to the medium (M. aquaticus) were not harmed by decompression from gas saturation pressures up to 300 atm.     

Septal pore cap protein SPC18, isolated from the basidiomycetous fungus Rhizoctonia solani, also resides in pore plugs

The hyphae of filamentous fungi are compartmentalized by septa that have a central pore. The fungal septa and septum-associated structures play an important role in maintaining cellular and intrahyphal homeostasis. The dolipore septa in the higher Basidiomycota (i.e., Agaricomycotina) are associated with septal pore caps. Although the ultrastructure of the septal pore caps has been studied extensively, neither the biochemical composition nor the function of these organelles is known. Here, we report the identification of the glycoprotein SPC18 that was found in the septal pore cap-enriched fraction of the basidiomycetous fungus Rhizoctonia solani. Based on its N-terminal sequence, the SPC18 gene was isolated. SPC18 encodes a protein of 158 amino acid residues, which contains a hydrophobic signal peptide for targeting to the endoplasmic reticulum and has an N-glycosylation motif. Immunolocalization showed that SPC18 is present in the septal pore caps. Surprisingly, we also observed SPC18 being localized in some plugs. The data reported here strongly support the hypothesis that septal pore caps are derived from endoplasmic reticulum and are involved in dolipore plugging and, thus, contribute to hyphal homeostasis in basidiomycetous fungi.     

Pbp2x localizes separately from Pbp2b and other peptidoglycan synthesis proteins during later stages of cell division of Streptococcus pneumoniae D39

The relative localization patterns of class B penicillin‐binding proteins Pbp2x and Pbp2b were used as positional indicators of septal and peripheral (side‐wall‐like) peptidoglycan (PG) synthesis, respectively, in the mid‐cell regions of Streptococcus pneumoniae cells at different stages of division. We confirm that Pbp2x and Pbp2b are essential in the strain D39 genetic background, which differs from that of laboratory strains. We show that Pbp2b, like Pbp2x and class A Pbp1a, follows a different localization pattern than FtsZ and remains at division septa after FtsZ reappears at the equators of daughter cells. Pulse‐experiments with fluorescent D‐amino acids (FDAAs) were performed in wild‐type cells and in cells in which Pbp2x activity was preferentially inhibited by methicillin or Pbp2x amount was depleted. These experiments show that Pbp2x activity separates from that of other PBPs to the centres of constricting septa in mid‐to‐late divisional cells resolved by high‐resolution 3D‐SIM microscopy. Dual‐protein and protein‐fluorescent vancomycin 2D and 3D‐SIM immunofluorescence microscopy (IFM) of cells at different division stages corroborate that Pbp2x separates to the centres of septa surrounded by an adjacent constricting ring containing Pbp2b, Pbp1a and regulators, StkP and MreC. The separate localization of Pbp2x suggests distinctive roles in completing septal PG synthesis and remodelling.