Septation Sites Research Articles

Lytic transglycosylase Slt of Pseudomonas aeruginosa as a periplasmic hub protein.

Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber-codon-suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber-codon-suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass-spectrometry-based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (KD < 0.5 μM) include PBPs (PBP1a, KD = 0.07 μM; PBP5 = 0.4 μM); other lytic transglycosylases (SltB2, KD = 0.09 μM; RlpA, KD = 0.4 μM); a type VI secretion system effector (Tse5, KD = 0.3 μM); and a regulatory protease for alginate biosynthesis (AlgO, KD < 0.4 μM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm.

Spatio-temporal control of asymmetric septum positioning during sporulation in Bacillus subtilis

During sporulation, Bacillus subtilis forms an asymmetric septum, dividing the cell into two compartments, a mother cell and a forespore. The site of asymmetric septation is linked to the membrane where FtsZ and SpoIIE initiate the formation of the Z-ring and the E-ring, respectively. These rings then serve as a scaffold for the other cell division and peptidoglycan synthesizing proteins needed to build the septum. However, despite decades of research, not enough is known about how the asymmetric septation site is determined. Here, we identified and characterized the interaction between SpoIIE and RefZ. We show that these two proteins transiently co-localize during the early stages of asymmetric septum formation when RefZ localizes primarily from the mother cell side of the septum. We propose that these proteins and their interplay with the spatial organization of the chromosome play a role in controlling asymmetric septum positioning.

Nucleotide-dependent activities of FtsA regulate the early establishment of a functional divisome during the Escherichia coli cell cycle.

During cell division in Escherichia coli, the highly conserved tubulin homolog FtsZ polymerizes and assembles into a ring-like structure, called the Z-ring, at the site of septation. For recruitment to the membrane surface, FtsZ polymers directly interact with membrane-associated proteins, predominantly FtsA in E. coli. FtsA shares structural homology with actin and, like actin, hydrolyzes ATP. Yeast actin detects nucleotide occupancy through a sensor region adjacent to the nucleotide binding site and adopts distinct conformations in monomeric and filamentous actin. Bacterial actin homologs also display considerable conformational flexibility across different nucleotide-bound states and polymerize. Here, we show that several amino acid residues proximal to the nucleotide binding site in FtsA are critical for function in vitro and in vivo. Each of these residues are important for ATP hydrolysis, phospholipid (PL) binding, ATP-dependent vesicle remodeling, and recruitment to the divisome in vivo, to varying degrees. Notably, we observed that Ser 84 and Glu 14 are essential for ATP-dependent vesicle remodeling and magnesium-dependent membrane release of FtsA from vesicles in vitro, and these defects likely underlie the loss of function by FtsA(E14R) and FtsA(S84L) in vivo. Finally, we demonstrate that FtsA(A188V), which is associated with temperature-sensitive growth in vivo, is defective for rapid ATP hydrolysis and ATP-dependent remodeling of PL vesicles in vitro. Together, our results show that loss of nucleotide-dependent activities by FtsA, such as ATP hydrolysis, membrane binding and release, and, most importantly, ATP-dependent PL remodeling, lead to failed Z-ring assembly and division defects in cells.

Exploring the interface between the first and second heart fields: Implications for cardiac septation

The embryonic heart elongates by progressive addition of second heart field (SHF) cardiac progenitor cells to the arterial and venous poles of the early heart tube, derived from the first heart field (FHF). The SHF gives rise to the right ventricle, outflow tract and part of the atrial myocardium. SHF-derived parts of the heart are hotspots of congenital heart defects (CHD) and perturbation of SHF deployment underlies a spectrum of CHD. These include atrial and ventricular septal defects that account for over 50% of CHD. Atrial septal structures originate in Tbx1 expressing cells in the posterior dorsal pericardial wall that activate the FHF regulator Tbx5 in response to retinoic acid (RA) signaling. Transient coexpression of Tbx1 and Tbx5 is followed by downregulation of the SHF program, and emergence of a sharp boundary between arterial and venous pole progenitor cells. A similar history of expression of both FHF and SHF regulators, followed by downregulation of the SHF program, demarcates boundary formation at the site of ventricular septation. Here we investigate the cellular and molecular mechanisms of ventricular septum morphogenesis. Using mouse genetics and a conditional dominant negative RA receptor, we show that RA signaling is also required at the heart field interface for morphogenesis of the muscular ventricular septum. Blocking RA signal reception in the SHF results in a bifid ventricular phenotype, identifying a novel RA-dependent zipper phase of ventricular septal morphogenesis. Additionally, blocking RA signal reception in the SHF results in conotruncal CHD. Together, our results point to the importance of RA signaling at the heart field interface for septal morphogenesis, providing new insights into the etiology of CHD.

Synthetic Assembly of Bacterial Divisome Machinery in Vitro

Bacterial cells grow and reproduce through coordination of many proteins collectively known as the cell division machinery. This machinery facilitates the division and physical separation of one cell into two identical daughter cells. The components of this cell division machinery are similar between bacterial taxa. In Escherichia coli, the cytoskeletal protein FtsZ promotes the formation of a division septum at the cell center and assembles into a large protein structure called the Z‐ring. FtsZ protomers in the Z‐ring form large linear polymers that assemble in a head to tail arrangement. Protein interactions between FtsZ in the Z‐ring and additional cell division proteins direct peptidoglycan synthesis and facilitate septation of the cell. FtsN, which may trigger septation, is a bitopic membrane protein with a small cytoplasmic domain and large periplasmic domain containing a peptidoglycan‐binding SPOR domain. FtsN interacts with other cell division proteins on both sides of the cytoplasmic membrane. In the cytoplasm, FtsN interacts with FtsA, an actin homolog, which tethers FtsZ polymers to the membrane. In the periplasm FtsN interacts with the FtsQBL complex and may help to recruit peptidoglycan synthetases such as FtsI. One proposed linear mechanism for activating cell wall synthesis begins with FtsA, which binds to FtsN in the cytoplasm, and causes FtsN to relay information to proteins in the periplasm, such as FtsQBL, to begin remodeling of the cell wall. Thus, it is critical that FtsN localizes to the future site of septation in order to carry out this function. Here we use purified proteins, including FtsZ, FtsA, and FtsN, to reconstitute the membrane‐spanning division complex in vitro and assemble a synthetic divisome scaffold. This reconstituted divisome subassembly allows us to probe direct protein interactions, protein conformations, assembly architectures, and nucleotide requirements in the cell division pathway, thus providing novel insight into the mechanics of division.

The Aspergillus nidulans IQGAP orthologue SepG is required for constriction of the contractile actomyosin ring

In this research we report that the sepG1 mutation in Aspergillus nidulans resides in gene AN9463, which is predicted to encode an IQGAP orthologue. The genetic lesion is predicted to result in a G-to-R substitution at residue 1637 of the 1737-residue protein in a highly conserved region of the RasGAP-C-terminal (RGCT) domain. When grown at restrictive temperature, strains expressing the sepGG1637R (sepG1) allele are aseptate, with reduced colony growth and aberrantly formed conidiophores. The aseptate condition can be replicated by deletion of AN9463 or by downregulating its expression via introduced promoters. The mutation does not prevent assembly of a cortical contractile actomyosin ring (CAR) at putative septation sites, but tight compaction of the rings is impaired and the rings fail to constrict. Both GFP::SepG wild type and the GFP-tagged product of the sepG1 allele localize to the CAR at both permissive and restrictive temperatures. Downregulation of myoB (encoding the A. nidulans type-II myosin heavy chain) does not prevent formation of SepG rings at septation sites, but filamentous actin is required for CAR localization of SepG and MyoB. We identify fourteen probable IQ-motifs (EF-hand protein binding sites) in the predicted SepG sequence. Two of the A. nidulans EF-hand proteins, myosin essential light chain (AnCdc4) and myosin regulatory light chain (MrlC), colocalize with SepG and MyoB at all stages of CAR formation and constriction. However, calmodulin (CamA) appears at septation sites only after the CAR has become fully compacted. When expression of sepG is downregulated, leaving MyoB as the sole IQ-motif protein in the pre-compaction CAR, both MrlC and AnCdc4 continue to associate with the forming CAR. When myoB expression is downregulated, leaving SepG as the sole IQ-motif protein in the CAR, AnCdc4 association with the forming CAR continues but MrlC fails to associate. This supports a model in which the IQ motifs of MyoB bind both MrlC and AnCdc4, while the IQ motifs of SepG bind only AnCdc4. Downregulation of either mrlC or Ancdc4 results in an aseptate phenotype, but has no effect on association of either SepG or MyoB with the CAR.

The Scaffold Proteins Paxillin B and α-Actinin Regulate Septation in Aspergillus nidulans via Control of Actin Ring Contraction.

Cytokinesis, as the final step of cell division, plays an important role in fungal growth and proliferation. In the filamentous fungus Aspergillus nidulans, defective cytokinesis is able to induce abnormal multinuclear or nonnucleated cells and then result in reduced hyphal growth and abolished sporulation. Previous studies have reported that a conserved contractile actin ring (CAR) protein complex and the septation initiation network (SIN) signaling kinase cascade are required for cytokinesis and septation; however, little is known about the role(s) of scaffold proteins involved in these two important cellular processes. In this study, we show that a septum-localized scaffold protein paxillin B (PaxB) is essential for cytokinesis/septation in A. nidulans. The septation defects observed in a paxB deletion strain resemble those caused by the absence of another identified scaffold protein, α-actinin (AcnA). Deletion of α-actinin (AcnA) leads to undetectable PaxB at the septation site, whereas deletion of paxB does not affect the localization of α-actinin at septa. However, deletion of either α-actinin (acnA) or paxB causes the actin ring to disappear at septation sites during cytokinesis. Notably, overexpression of α-actinin acnA partially rescues the septum defects of the paxB mutant but not vice versa, suggesting AcnA may play a dominant role over that of PaxB for cytokinesis and septation. In addition, PaxB and α-actinin affect the septal dynamic localization of MobA, a conserved component of the SIN pathway, suggesting they may affect the SIN protein complex function at septa. Protein pull-down assays combined with liquid chromatography–mass spectrometry identification indicate that α-actinin AcnA and PaxB likely do not directly interact, but presumably belong to an actin cytoskeleton protein network that is required for the assembly and contraction of the CAR. Taken together, findings in this study provide novel insights into the roles of conserved scaffold proteins during fungal septation in A. nidulans.

The Bacterial Tubulin Homolog FtsZ Forms 2D-sheets that Sustain Electrical Oscillations

FtsZ, a major cytoskeletal protein present in all bacteria and archaea forms a cytokinetic ring, the Z ring, at the site of septation, which directs cytokinesis. Multiple sequence alignment and secondary structure predictions indicate that bacterial FtsZ is a homolog of the eukaryotic tubulins which, as their mammalian counterpart, binds guanine nucleotides, has GTPase activity, and is able to assemble into protofilamental structures in a GTP-dependent manner. Previous studies from our laboratory demonstrated that microtubule (MTs) structures isolated from murine, bovine and apian brains spontaneously generate electrical oscillations and bursts of electrical activity similar to action potentials. Although the electrical behavior of brain MTs may have important implication in eukaryotic cell function, no information is available as to whether FtsZ may share similar properties. Therefore, the aim of the present study was to explore whether FtsZ protofilamental sheets may sustain electrical activity. FtsZ protein from E. coli ATCC 25922 was purified by ammonium sulfate precipitation, and assembled into protofilamental sheets by incubation with a polymerization buffer. 2D FtsZ sheets were voltage-clamped, and gigaseals obtained in most experiments (n = 7). Subsequently, electrical recordings from output currents were obtained, showing a complex oscillatory behavior with several peak frequencies between 7 and 110 Hz in the power spectra. As a result, it was observed that FtsZ 2D sheets had a much wider range of frequencies compared to MTs sheets from eukaryotic origin. The findings demonstrate that FtsZ protofilamental sheets are capable of generating electrical signals and behave as electrical oscillators that may be relevant to the cell division process and/or yet unknown signaling mechanisms.

Analyzing Protein Kinase C Interactions With Rho‐type GTPases in the Filamentous Fungus Aspergillus nidulans

Cell division in filamentous fungi involves coalescence and subsequent constriction of a contractile actomyosin ring (CAR), as in other fungi and metazoans. In fungi, constriction of the CAR is followed by deposition of cell wall material resulting in a septum. In the model organism, Aspergillus nidulans, many proteins are involved in septation including protein kinase C (PkcA), the formin SepA, and the GTPase Rho4. The current study focuses on the relationships among PkcA, SepA, and Rho‐type GTPases during septation. PkcA contains two putative N‐terminal Rho binding domains designated HR1A and HR1B. The formin SepA belongs to the Diaphanous‐related family of formins that contain an N‐terminal Rho binding domain that upon binding of the appropriate Rho GTPase results in formin activation. Reports from another group suggest that in A. nidulans Rho4 is likely the GTPase required for SepA activation. In previous work we showed that PkcA both physically and functionally interacts with SepA via one of its three formin homology domains (FH2), and in the sepA1 temperature sensitive mutant (FH2 domain‐L1369S) which is aseptate at elevated temperature, PkcA fails to localize to septation sites at elevated temperature. Here we report via yeast two‐hybrid assay that PkcA interacts with SepA via one of its putative Rho binding domains (HR1A), and that expressing extra copies of pkcA in a rho4‐null mutant increased colony diameter and improved sporulation. PkcA localization to septation sites is reduced in a rhoA mutant while septation is unaffected. The spatiotemporal relationship among PkcA, SepA, Rho4, and RhoA will be further investigated via yeast two‐hybrid, bimolecular complementation assays, and localization of fluorescence labeled proteins in mutant strains.Support or Funding InformationSupport for this work was provided by the National Science Foundation Grant 1615192 (Loretta Jackson‐Hayes, PI).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Asymmetric cell division during Bacillus subtilis sporulation.

Bacillus subtilis is a rod-shaped bacterium which divides precisely at mid-cell during vegetative growth. Unlike Escherichia coli, another model organism used for studying cell division, B. subtilis can also divide asymmetrically during sporulation, the simplest cell differentiation process. The asymmetrically positioned sporulation septum serves as a morphological foundation for establishing differential gene expression in the smaller forespore and larger mother cell. Both vegetative and sporulation septation events are fine-tuned with cell cycle, and placement of both septa are highly precise. We understand in some detail how this is achieved during vegetative growth but have limited information about how the asymmetric septation site is determined during sporulation.

Aspergillus nidulans protein kinase C forms a complex with the formin SepA that is involved in apical growth and septation

The Aspergillus nidulans orthologue of Protein kinase C (PkcA) and the A. nidulans formin SepA participate in polarized growth. PkcA localizes to growing hyphal apices and septation sites, and amino acid sequences within PkcA that are required for PkcA to localize to these sites of cell wall synthesis have been identified. SepA is associated with the contractile actomyosin ring (CAR), and it localizes at hyphal tips in association with the Spitzenkörper (SPK) and as an apical dome. A mutation in the sepA gene (sepA1) renders A. nidulans aseptate at elevated temperature. Progress towards understanding the spatiotemporal relationship between PkcA and SepA during polarized growth is presented here. Fluorescent chimeras of PkcA and SepA strongly overlapped in some hyphal tips in a dome pattern, while other tips displayed SepA SPK and PkcA dome localization within the same tip. At septation sites PkcA and SepA consistently colocalized through late stages of CAR constriction. Bimolecular fluorescence complementation experimental results provide evidence that SepA and PkcA are both present in complexes at both hyphal tip domes and at cortical rings. A Gal4-based yeast two-hybrid analysis confirmed the physical interaction between SepA and PkcA, and indicted that the FH2 domain of SepA is involved in its physical interaction with PkcA. A functional interaction between PkcA and SepA was shown through complementation of the pkcA calC2 mutant’s hypersensitivity to cell wall perturbing agents by overexpressed sepA and by the ability of the sepA1 mutation to block PkcA’s ability to form cortical rings. Taken together these results suggest that a PkcA/SepA complex is involved in polarized growth. Through experiments using the actin disrupter latrunculin B, evidence is presented suggesting that actin plays a role in the PkcA/SepA complex.

UmTea1, a Kelch and BAR domain-containing protein, acts at the cell cortex to regulate cell morphogenesis in the dimorphic fungus Ustilago maydis

The spatial organization of a cell is crucial for distribution of cell components and for cell morphogenesis in all organisms. Ustilago maydis, a basidiomycete fungus, has a yeast-like and a filamentous form. The former buds once per cell cycle at one of the cell poles, and can use the same site repeatedly or choose a new site at the same pole or opposite pole. The filamentous form consists of a long apical cell with short septate basal compartments lacking cytoplasm. It grows at the apex and can reverse growth forming a new growth zone at the basal end. We are interested in understanding how these different morphologies are generated. Here we present identification and characterization of U. maydis Tea1, a homologue of the fission yeast cell end marker Tea1. We demonstrate that UmTea1, a Kelch domain protein, interacts with itself and is an important determinant of the site of polarized growth: tea1 mutants bud simultaneously from both cell poles and form bifurcate buds. UmTea1 also regulates septum positioning, cell wall deposition, cell and neck width, coordination of nuclear division and cell separation, and localization of sterol-rich membrane domains. Some of these functions are shared with UmTea4, another cell end marker. We show that Tea1::GFP localizes to sites of polarized or potential polarized growth and to the septation site in the yeast-like form. Additionally, localization of Tea1::GFP as rings along the filament suggests that the filament undergoes septation. We hypothesize that Tea1 may act as a scaffold for the assembly of proteins that determine the site of polarized growth.

The positioning of the asymmetric septum during sporulation in Bacillus subtilis.

Probably one of the most controversial questions about the cell division of Bacillus subtilis, a rod-shaped bacterium, concerns the mechanism that ensures correct division septum placement–at mid-cell during vegetative growth but closer to one end during sporulation. In general, bacteria multiply by binary fission, in which the division septum forms almost exactly at the cell centre. How the division machinery achieves such accuracy is a question of continuing interest. We understand in some detail how this is achieved during vegetative growth in Escherichia coli and B. subtilis, where two main negative regulators, nucleoid occlusion and the Min system, help to determine the division site, but we still do not know exactly how the asymmetric septation site is determined during sporulation in B. subtilis. Clearly, the inhibitory effects of the nucleoid occlusion and Min system on polar division have to be overcome. We evaluated the positioning of the asymmetric septum and its accuracy by statistical analysis of the site of septation. We also clarified the role of SpoIIE, RefZ and MinCD on the accuracy of this process. We determined that the sporulation septum forms approximately 1/6 of a cell length from one of the cell poles with high precision and that SpoIIE, RefZ and MinCD have a crucial role in precisely localizing the sporulation septum. Our results strongly support the idea that asymmetric septum formation is a very precise and highly controlled process regulated by a still unknown mechanism.

Analyzing Protein Kinase C Domain Interactions with the Formin SepA

This research focuses on understanding polarized growth in filamentous fungi. In the model organism Aspergillus nidulans and other filamentous fungi, polarized growth is focused at both the hyphal tip and septa, and growth at both areas involves deposition of cell wall material. To better understand polarized growth, we study proteins that localize to growing hyphal tips and forming septa. In A. nidulans, protein kinase C (PkcA) is involved in the organism's cell wall stress response, and our previous work uncovered a protein‐protein interaction between PkcA and the formin SepA. We also illustrated complementary roles of the two proteins in growth and cell wall integrity. Both PkcA and SepA localize to hyphal tips and septation sites. Using bimolecular fluorescence complementation, we found that SepA and PkcA physically interact at both sub‐cellular locations, and that this interaction occurs through amino acid residues within the C‐terminal half of SepA. Previously, we showed using a series of fluorescence‐labeled PkcA truncated proteins that two regions of PkcA are responsible for PkcA localizing to hyphal tips and septation sites. The first region is a 10 amino‐acid sequence near the carboxyl end of the C2 domain that is required for localization to hyphal tips and septation sites, while the second region is between the C2 and C1B domain, and is sufficient for localization to septation sites only. Here we present our progress towards determining which PkcA domains are involved in its interaction with SepA by using the previously published PkcA truncations in Gal4‐based yeast two hybrid assays. These results help to shed light on the mechanism underlying SepA's and PkcA's complementary involvement in polarized growth.Support or Funding InformationNational Science Foundation Grant 1615192 (Loretta Jackson‐Hayes, PI)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Analyzing Protein Kinase C Interactions with Rho4 and Bud3 in the Filamentous Fungus Aspergillus nidulans

Cell division in filamentous fungi involves coalescence and subsequent constriction of a contractile actomyosin ring (CAR), as in other fungi and metazoans. In fungi, constriction of the CAR is followed by deposition of cell wall material resulting in a septum. In the model organism, Aspergillus nidulans, many proteins are involved in septation including protein kinase C (PkcA), the formin SepA, the GTPase Rho4, and its guanine nucleotide exchange factor (GEF) called Bud3. In previous work we showed that PkcA both physically and functionally interacts with SepA. The formin SepA belongs to the Diaphanous‐related family of formins that contain an N‐terminal Rho binding domain that upon binding of the appropriate Rho GTPase results in formin activation. In A. nidulans Rho4 is likely the GTPase required for SepA activation. Reports from another group have shown that Rho4 and Bud3 work as a module that is required for septation to occur. The current study focuses on the relationship of the Bud3/Rho4 partnership with PkcA and SepA during polarized growth. Using bimolecular fluorescence complementation (BiFC) and overexpressing PkcA in Bud3‐ and Rho4‐null strains, we examined the relationships between PkcA and Rho4 and PkcA and Bud3. Through BiFC we found that PkcA and Rho4 interact at septation sites. Overexpressing PkcA in a Rho4‐null mutant strain increased colony diameter and improved sporulation. However, PkcA overexpression in a Bud3‐null mutant had no effect on growth and sporulation. These results suggest that there are physical and functional interactions between PkcA and Rho4 that are important in polarized growth.Support or Funding InformationNational Science Foundation Grant 1615192 (Loretta Jackson‐Hayes, PI)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Life without Division: Physiology of Escherichia coli FtsZ-Deprived Filaments.

ABSTRACTWhen deprived of FtsZ, Escherichia coli cells (VIP205) grown in liquid form long nonseptated filaments due to their inability to assemble an FtsZ ring and their failure to recruit subsequent divisome components. These filaments fail to produce colonies on solid medium, in which synthesis of FtsZ is induced, upon being diluted by a factor greater than 4. However, once the initial FtsZ levels are recovered in liquid culture, they resume division, and their plating efficiency returns to normal. The potential septation sites generated in the FtsZ-deprived filaments are not annihilated, and once sufficient FtsZ is accumulated, they all become active and divide to produce cells of normal length. FtsZ-deprived cells accumulate defects in their physiology, including an abnormally high number of unsegregated nucleoids that may result from the misplacement of FtsK. Their membrane integrity becomes compromised and the amount of membrane proteins, such as FtsK and ZipA, increases. FtsZ-deprived cells also show an altered expression pattern, namely, transcription of several genes responding to DNA damage increases, whereas transcription of some ribosomal or global transcriptional regulators decreases. We propose that the changes caused by the depletion of FtsZ, besides stopping division, weaken the cell, diminishing its resiliency to minor challenges, such as dilution stress.

Activation of Xer-recombination at dif: structural basis of the FtsKγ-XerD interaction.

Bacterial chromosomes are most often circular DNA molecules. This can produce a topological problem; a genetic crossover from homologous recombination results in dimerization of the chromosome. A chromosome dimer is lethal unless resolved. A site-specific recombination system catalyses this dimer-resolution reaction at the chromosomal site dif. In Escherichia coli, two tyrosine-family recombinases, XerC and XerD, bind to dif and carry out two pairs of sequential strand exchange reactions. However, what makes the reaction unique among site-specific recombination reactions is that the first step, XerD-mediated strand exchange, relies on interaction with the very C-terminus of the FtsK DNA translocase. FtsK is a powerful molecular motor that functions in cell division, co-ordinating division with clearing chromosomal DNA from the site of septation and also acts to position the dif sites for recombination. This is a model system for unlinking, separating and segregating large DNA molecules. Here we describe the molecular detail of the interaction between XerD and FtsK that leads to activation of recombination as deduced from a co-crystal structure, biochemical and in vivo experiments. FtsKγ interacts with the C-terminal domain of XerD, above a cleft where XerC is thought to bind. We present a model for activation of recombination based on structural data.

Cell wall protection by the Candida albicans class I chitin synthases

Candida albicans has four chitin synthases from three different enzyme classes which deposit chitin in the cell wall, including at the polarized tips of growing buds and hyphae, and sites of septation. The two class I enzymes, Chs2 and Chs8, are responsible for most of the measurable chitin synthase activity in vitro, but their precise biological functions in vivo remain obscure. In this work, detailed phenotypic analyses of a chs2Δchs8Δ mutant have shown that C. albicans class I chitin synthases promote cell integrity during early polarized growth in yeast and hyphal cells. This was supported by live cell imaging of YFP-tagged versions of the class I chitin synthases which revealed that Chs2-YFP was localized at sites of polarized growth. Furthermore, a unique and dynamic pattern of localization of the class I enzymes at septa of yeast and hyphae was revealed. Phosphorylation of Chs2 on the serine at position 222 was shown to regulate the amount of Chs2 that is localized to sites of polarized growth and septation. Independently from this post-translational modification, specific cell wall stresses were also shown to regulate the amount of Chs2 that localizes to specific sites in cells, and this was linked to the ability of the class I enzymes to reinforce cell wall integrity during early polarized growth in the presence of these stresses.

Two amino acid sequences direct Aspergillus nidulans protein kinase C (PkcA) localization to hyphal apices and septation sites

The Aspergillus nidulans ortholog of protein kinase C (pkcA) is involved in the organism’s putative cell wall integrity (CWI) pathway, and PkcA also is highly localized at growing tips and forming septa. In the present work we identify the regions within PkcA that are responsible for its localization to hyphal tips and septation sites. To this end, we used serially truncated pkcA constructs and expressed them as green fluorescent protein (GFP) chimeras and identified two regions that direct PkcA localization. The first region is a 10 amino-acid sequence near the carboxyl end of the C2 domain that is required for localization to hyphal tips. Proteins containing this sequence also localize to septation sites. A second region between C2 and C1B (encompassing C1A) is sufficient for localization to septation sites but not to hyphal tips. We also report that localization to hyphal tips and septation sites alone is not sufficient for truncated constructs to complement hypersensitivity to the cell wall compromising agent calcofluor white in a strain bearing a mutation in the pkcA gene. Taken together, these results suggest that localization and stress response might be independent.

A mutation in the converter subdomain of Aspergillus nidulans MyoB blocks constriction of the actomyosin ring in cytokinesis

We have identified a mutant allele of the Aspergillus nidulans homologue of myosin II (myoB; AN4706), which prevents normal septum formation. This is the first reported myosin II mutation in a filamentous fungus. Strains expressing the myoBG843D allele produce mainly aberrant septa at 30°C and are completely aseptate at temperatures above 37°C. Conidium formation is greatly reduced at 30°C and progressively impaired with increasing temperature. Sequencing of the myoBG843D allele identified a point mutation predicted to result in a glycine-to-aspartate amino acid substitution at residue 843 in the myosin II converter domain. This residue is conserved in all fungal, plant, and animal myosin sequences that we have examined. The mutation does not prevent localization of the myoBG843D gene product to contractile rings, but it does block ring constriction. MyoBG843D rings at sites of abortive septation disassemble after an extended period and dissipate into the cytoplasm. During contractile ring formation, both wild type and mutant MyoB::GFP colocalize with actin – an association that begins at the pre-ring “string” stage. Down-regulation of wild-type myoB expression under control of the alcA promoter blocks septation but does not prevent actin from aggregating at putative septation sites – the actin rings, however, do not fully coalesce. Both septation and targeting of MyoB are blocked by disruption of filamentous actin using latrunculin B. We propose a model in which myosin assembly at septation sites depends upon the presence of F-actin, but assembly of the actin component of contractile rings depends upon normal levels of myosin only for the final stages of ring compaction.