The analysis of 13C-labeled lipids by mass spectrometry is challenging due to the complexity from labeling the large number of carbon atoms in lipids. To further add to the complexity, different adducts can be produced during electrospray ionization and in-source fragmentation, which can create complex overlapping isotope patterns that can only be resolved using high-resolution mass spectrometry. Co-elution of lipids even after chromatographic separation also adds to the potential for overlapping mass spectra. Here, we describe a procedure that enables full 13C-labeled patterns to be resolved in complex microalgal lipid extracts as well a procedure that provides structural labeling information. Mass resolving powers of 240000 full width half-maximum (fwhm) and fast targeted MS/MS allowed the differentiation of isotopologues, adducts, and unresolved lipid species after chromatographic separation. This enabled the percentage of 13C enrichment to be calculated for each individual lipid species over a time series in the microalgal lipidome. The application of tandem mass spectrometry (MS/MS) also allowed the degree of labeling within the headgroup vs acyl chains to be determined, further adding to the detail of information collected. This information is particularly useful for studying lipid synthesis and remodeling processes and can be extended to other biological systems.

Plant diseases caused by Phytophthora species are serious threats to agriculture and the natural environment. Genome sequencing has revealed the lack of a gene for canonical phospholipase C (PLC), an enzyme that was hitherto thought to be ubiquitous in eukaryotes. PLC acts in the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2 ), a membrane-bound phospholipid critical for signal initiation in many cellular processes. Previous studies have not provided evidence of endogenous PtdIns-4,5-P2 in Phytophthora and, in the absence of canonical PLC, argued for redundancy or loss in the PLC pathway in Phytophthora. Using liquid chromatography mass spectrometry, we have detected endogenous PtdIns-4,5-P2 in Phytophthora cinnamomi. This is the first identification of the phospholipid in the genus, and is significant because it indicates that the signal transduction pathway of the PLC product, inositol 1,4,5-trisphosphate (IP3 ), may have been retained in Phytophthora incorporating an as-yet unidentified homolog or analog of PLC.

Background: The oomycete plant pathogen, Phytophthora cinnamomi, is responsible for the destruction of thousands of species of native Australian plants, as well as several crops, such as avocado and macadamia, and has one of the widest host-plant ranges of the Phytophthora genus. The current reference genome of P. cinnamomi is based on an atypical strain and has large gaps in its assembly. To further studies of the pathogenicity of this species, especially in Australia, robust genome assemblies of more typical strains are required. Here we report the genome sequencing, draft assembly, and preliminary annotation of two geographically separated Australian strains of P. cinnamomi. Findings: Some 308 million raw reads were generated for the two strains, DU054 and WA94.26. Independent genome assembly produced final genome sequences of 62.8 Mb (in 14,268 scaffolds) and 68.1 Mb (in 10,084 scaffolds), which are comparable in size and contiguity to other Phytophthora genomes. Gene prediction yielded > 22,000 predicted protein-encoding genes within each genome, while BUSCO assessment showed 94.4% and 91.5% of the stramenopile single-copy orthologs to be present in the assembled genomes, respectively. Conclusions: The assembled genomes of two geographically distant isolates of Phytophthora cinnamomi will provide a valuable resource for further comparative analyses and evolutionary studies of this destructive pathogen, and further annotation of the presented genomes may yield possible targets for novel pathogen control methods.

Background: The oomycete plant pathogen, Phytophthora cinnamomi, is responsible for the destruction of thousands of species of native Australian plants, as well as several crops, such as avocado and macadamia, and has one of the widest host-plant ranges of the Phytophthora genus. The current reference genome of P. cinnamomi is based on an atypical strain and has large gaps in its assembly. To further studies of the pathogenicity of this species, especially in Australia, robust genome assemblies of more typical strains are required. Here we report the genome sequencing, draft assembly, and preliminary annotation of two geographically separated Australian strains of P. cinnamomi. Findings: Some 308 million raw reads were generated for the two strains, DU054 and WA94.26. Independent genome assembly produced final genome sequences of 62.8 Mb (in 14,268 scaffolds) and 68.1 Mb (in 10,084 scaffolds), which are comparable in size and contiguity to other Phytophthora genomes. Gene prediction yielded > 22,000 predicted protein-encoding genes within each genome, while BUSCO assessment showed 94.4% and 91.5% of the stramenopile single-copy orthologs to be present in the assembled genomes, respectively. Conclusions: The assembled genomes of two geographically distant isolates of Phytophthora cinnamomi will provide a valuable resource for further comparative analyses and evolutionary studies of this destructive pathogen, and further annotation of the presented genomes may yield possible targets for novel pathogen control methods.

SUMMARY Human patients with mitochondrial diseases are more susceptible to bacterial infections, particularly of the respiratory tract. To investigate the susceptibility of mitochondrially diseased cells to an intracellular bacterial respiratory pathogen, we exploited the advantages of Dictyostelium discoideum as an established model for mitochondrial disease and for Legionella pneumophila pathogenesis. Legionella infection of macrophages involves recruitment of mitochondria to the Legionella-containing phagosome. We confirm here that this also occurs in Dictyostelium and investigate the effect of mitochondrial dysfunction on host cell susceptibility to Legionella. In mitochondrially diseased Dictyostelium strains, the pathogen was taken up at normal rates, but it grew faster and reached counts that were twofold higher than in the wild-type host. We reported previously that other mitochondrial disease phenotypes for Dictyostelium are the result of the activity of an energy-sensing cellular alarm protein, AMP-activated protein kinase (AMPK). Here, we show that the increased ability of mitochondrially diseased cells to support Legionella proliferation is suppressed by antisense-inhibiting expression of the catalytic AMPKα subunit. Conversely, mitochondrial dysfunction is phenocopied, and intracellular Legionella growth is enhanced, by overexpressing an active form of AMPKα in otherwise normal cells. These results indicate that AMPK signalling in response to mitochondrial dysfunction enhances Legionella proliferation in host cells.

Laser scanning confocal microscopy and TEM were used to study the morphology of secondary plastids in algae of the genus Mallomonas (Synurophyceae). At interphase, Mallomonas splendens (G. S. West) Playfair, M. rasilis Dürrschm., M. striata Asmund, and M. adamas K. Harris et W. H. Bradley contained a single H‐shaped plastid consisting of two large lobes connected by a narrow isthmus. Labeling of DNA revealed a necklace‐like arrangement of plastid nucleoids at the periphery of the M. splendens plastid and a less‐patterned array in M. rasilis. The TEM of M. splendens and M. rasilis showed an electron‐dense belt surrounding the plastid isthmus in interphase cells; this putative plastid‐dividing ring (PD ring) was adpressed to the inner pair of the four plastid membranes, suggesting that it is homologous to the PD ring of green and red plastids. The PD ring did not contain actin (indicated by lack of staining with phalloidin) and displayed filaments or tubules of 5–10 nm in diameter that may be homologous to the tubules described in red algal PD rings. Confocal microscopy of chl autofluorescence from M. splendens showed that the plastid isthmus was severed as mitosis began, giving rise to two single‐lobed daughter plastids, which, as mitosis and cell division progressed, separated from one another and then each constricted to form the H‐shaped plastids of daughter cells. Similar plastid division cycles were observed in M. rasilis and M. adamas; however, the plastid isthmus of M. striata was retained throughout most of cell division and was eventually severed by the cell cleavage furrow.

Stomatin, originally identified as a major protein of the human erythrocyte membrane, is widely expressed in various tissues. Orthologues are found in vertebrates, invertebrates, plants, and microorganisms. Related proteins exhibit a common core structure, termed the prohibitin (PHB) domain, with varying extensions. Stomatin has an unusual topology, similar to caveolin-1, with a hydrophobic domain embedded at the cytoplasmic side of the membrane. Additional anchoring is provided by palmitoylation and the membrane affinity of the PHB domain. Stomatin associates with cholesterol-rich microdomains (lipid rafts), forms oligomers, and thereby displays a scaffolding function by generating large protein-lipid complexes. It regulates the activity of various membrane proteins by reversibly recruiting them to lipid rafts. This mechanism of regulation has been shown for GLUT-1 and may also apply for ion channels. Stomatin is located at the plasma membrane, particularly in microvilli, in endocytic and exocytic vesicles, and cytoplasmic granules. Stomatin-carrying endosomes are highly dynamic and interact with lipid droplets suggesting a role in intracellular lipid transport. This subcellular distribution and the caveolin-like protein structure suggest important membrane organizing functions for stomatin. A general picture emerges now that cell membranes contain cholesterol-rich domains that are generated and regulated by scaffolding proteins like caveolins, stomatins, and flotillin/reggie proteins.

Review(s) of: Climate Change: Turning up the Heat, by A Barrie Pittock, Publishers: CSIRO Publications, Collingwood; Earthscan, London, 2005. 316 pages, paperback; ISBN, RRP $39.95.

ABSTRACT The earliest stage in bacterial cell division is the formation of a ring, composed of the tubulin-like protein FtsZ, at the division site. Tight spatial and temporal regulation of Z-ring formation is required to ensure that division occurs precisely at midcell between two replicated chromosomes. However, the mechanism of Z-ring formation and its regulation in vivo remain unresolved. Here we identify the defect of an interesting temperature-sensitive ftsZ mutant (ts1) of Bacillus subtilis. At the nonpermissive temperature, the mutant protein, FtsZ(Ts1), assembles into spiral-like structures between chromosomes. When shifted back down to the permissive temperature, functional Z rings form and division resumes. Our observations support a model in which Z-ring formation at the division site arises from reorganization of a long cytoskeletal spiral form of FtsZ and suggest that the FtsZ(Ts1) protein is captured as a shorter spiral-forming intermediate that is unable to complete this reorganization step. The ts1 mutant is likely to be very valuable in revealing how FtsZ assembles into a ring and how this occurs precisely at the division site.

ABSTRACT To investigate protein import into the mitochondria of Dictyostelium discoideum , green fluorescent protein (GFP) was fused as a reporter protein either to variable lengths of the N-terminal region of chaperonin 60 (the first 23, 40, 80, 97, and 150 amino acids) or to the mitochondrial targeting sequence of DNA topoisomerase II. The fusion proteins were expressed in AX2 cells under the actin-15 promoter. Fluorescence images of GFP transformants confirmed that Dictyostelium chaperonin 60 is a mitochondrial protein. The level of the mitochondrially targeted GFP fusion proteins was unexpectedly much lower than the nontargeted (cytoplasmic) forms. The distinction between targeted and nontargeted protein activities was investigated at both the transcriptional and translational levels in vivo. We found that targeting GFP to the mitochondria results in reduced levels of the fusion protein even though transcription of the fusion gene and the stability of the protein are unaffected. [ 35 S]methionine labeling and GFP immunoprecipitation confirmed that mitochondrially targeted GFP is translated at much slower rates than nontargeted GFP. The results indicate a novel phenomenon, import-associated translational inhibition, whereby protein import into the mitochondria limits the rate of translation. The simplest explanation for this is that import of the GFP fusion proteins occurs cotranslationally, i.e., protein synthesis and import into mitochondria are coupled events. Consistent with cotranslational import, Northern analysis showed that the GFP mRNA is associated with isolated mitochondria. This association occurred regardless of whether the GFP was fused to a mitochondrial leader peptide. However, the presence of an import-competent leader peptide stabilized the mRNA-mitochondria association, rendering it more resistant to extensive EDTA washing. In contrast with GFP, the mRNA of another test protein, aequorin, did not associate with the mitochondria, and its translation was unaffected by import of the encoded polypeptide into the mitochondria.

Mitochondria are the product of an ancient endosymbiotic event between an alpha-proteobacterium and an archael host. An early barrier to overcome in this relationship was the control of the-bacterium's proliferation within the host. Undoubtedly, the bacterium (or protomitochondrion) would have used its own cell division apparatus to divide at first and, today a remnant of this system remains in some "ancient" and diverse eukaryotes such as algae and amoebae, the most conserved and widespread of all bacterial division proteins, FtsZ. In many of the eukaryotes that still use FtsZ to constrict the mitochondria from the inside, the mitochondria still resemble bacteria in shape and size. Eukaryotes, however, have a mitochondrial morphology that is often highly fluid, and in their tubular networks of mitochondria, division is clearly complemented by mitochondrial fusion. FtsZ is no longer used by these complex eukaryotes, and may have been replaced by other proteins better suited to sustaining complex mitochondrial networks. Although proteins that divide mitochondria from the inside are just beginning to be characterized in higher eukaryotes, many division proteins are known to act on the outside of the organelle. The most widespread of these are the dynamin-like proteins, which appear to have been recruited very early in the evolution of mitochondria. The essential nature of mitochondria dictates that their loss is intolerable to human cells, and that mutations disrupting mitochondrial division are more likely to be fatal than result in disease. To date, only one disease (Charcot-Marie-Tooth disease 2A) has been mapped to a gene that is required for mitochondrial division, whereas two other diseases can be attributed to mutations in mitochondrial fusion genes. Apart from playing a role in regulating the morphology, which might be important for efficient ATP production, research has indicated that the mitochondrial division and fusion proteins can also be important during apoptosis; mitochondrial fragmentation is an early triggering (and under many stimuli, essential) step in the pathway to cell suicide.

During spore formation in Bacillus subtilis, cell division occurs at the cell pole and is believed to require essentially the same division machinery as vegetative division. Intriguingly, although the cell division protein DivIB is not required for vegetative division at low temperatures, it is essential for efficient sporulation under these conditions. We show here that at low temperatures in the absence of DivIB, formation of the polar septum during sporulation is delayed and less efficient. Furthermore, the polar septa that are complete are abnormally thick, containing more peptidoglycan than a normal polar septum. These results show that DivIB is specifically required for the efficient and correct formation of a polar septum. This suggests that DivIB is required for the modification of sporulation septal peptidoglycan, raising the possibility that DivIB either regulates hydrolysis of polar septal peptidoglycan or is a hydrolase itself. We also show that, despite the significant number of completed polar septa that form in this mutant, it is unable to undergo engulfment. Instead, hydrolysis of the peptidoglycan within the polar septum, which occurs during the early stages of engulfment, is incomplete, producing a similar phenotype to that of mutants defective in the production of sporulation-specific septal peptidoglycan hydrolases. We propose a role for DivIB in sporulation-specific peptidoglycan remodelling or its regulation during polar septation and engulfment.

Mitochondrial fission requires the division of both the inner and outer mitochondrial membranes. Dynamin-related proteins operate in division of the outer membrane of probably all mitochondria, and also that of chloroplasts - organelles that have a bacterial origin like mitochondria. How the inner mitochondrial membrane divides is less well established. Homologues of the major bacterial division protein, FtsZ, are known to reside inside mitochondria of the chromophyte alga Mallomonas, a red alga, and the slime mould Dictyostelium discoideum, where these proteins are likely to act in division of the organelle. Mitochondrial FtsZ is, however, absent from the genomes of higher eukaryotes (animals, fungi, and plants), even though FtsZs are known to be essential for the division of probably all chloroplasts. To begin to understand why higher eukaryotes have lost mitochondrial FtsZ, we have sampled various diverse protists to determine which groups have retained the gene. Database searches and degenerate PCR uncovered genes for likely mitochondrial FtsZs from the glaucocystophyte Cyanophora paradoxa, the oomycete Phytophthora infestans, two haptophyte algae, and two diatoms - one being Thalassiosira pseudonana, the draft genome of which is now available. From Thalassiosira we also identified two chloroplast FtsZs, one of which appears to be undergoing a C-terminal shortening that may be common to many organellar FtsZs. Our data indicate that many protists still employ the FtsZ-based ancestral mitochondrial division mechanism, and that mitochondrial FtsZ has been lost numerous times in the evolution of eukaryotes.

ABSTRACT In bacteria, the protein FtsZ is the principal component of a ring that constricts the cell at division. Though all mitochondria probably arose through a single, ancient bacterial endosymbiosis, the mitochondria of only certain protists appear to have retained FtsZ, and the protein is absent from the mitochondria of fungi, animals, and higher plants. We have investigated the role that FtsZ plays in mitochondrial division in the genetically tractable protist Dictyostelium discoideum , which has two nuclearly encoded FtsZs, FszA and FszB, that are targeted to the inside of mitochondria. In most wild-type amoebae, the mitochondria are spherical or rod-shaped, but in fsz -null mutants they become elongated into tubules, indicating that a decrease in mitochondrial division has occurred. In support of this role in organelle division, antibodies to FszA and FszA-green fluorescent protein (GFP) show belts and puncta at multiple places along the mitochondria, which may define future or recent sites of division. FszB-GFP, in contrast, locates to an electron-dense, submitochondrial body usually located at one end of the organelle, but how it functions during division is unclear. This is the first demonstration of two differentially localized FtsZs within the one organelle, and it points to a divergence in the roles of these two proteins.

Integral proteins in the outer membrane of mitochondria control all aspects of organelle biogenesis, being required for protein import, mitochondrial fission, and, in metazoans, mitochondrial aspects of programmed cell death. How these integral proteins are assembled in the outer membrane had been unclear. In bacteria, Omp85 is an essential component of the protein insertion machinery, and we show that members of the Omp85 protein family are also found in eukaryotes ranging from plants to humans. In eukaryotes, Omp85 is present in the mitochondrial outer membrane. The gene encoding Omp85 is essential for cell viability in yeast, and conditional omp85 mutants have defects that arise from compromised insertion of integral proteins like voltage-dependent anion channel (VDAC) and components of the translocase in the outer membrane of mitochondria (TOM) complex into the mitochondrial outer membrane.