Repair deficient mice reveal mABH2 as the primary oxidative demethylase for repairing 1meA and 3meC lesions in DNA

Two human homologs of the Escherichia coli AlkB protein, denoted hABH2 and hABH3, were recently shown to directly reverse 1-methyladenine (1meA) and 3-methylcytosine (3meC) damages in DNA. We demonstrate that mice lacking functional mABH2 or mABH3 genes, or both, are viable and without overt phenotypes. Neither were histopathological changes observed in the gene-targeted mice. However, in the absence of any exogenous exposure to methylating agents, mice lacking mABH2, but not mABH3 defective mice, accumulate significant levels of 1meA in the genome, suggesting the presence of a biologically relevant endogenous source of methylating agent. Furthermore, embryonal fibroblasts from mABH2-deficient mice are unable to remove methyl methane sulfate (MMS)-induced 1meA from genomic DNA and display increased cytotoxicity after MMS exposure. In agreement with these results, we found that in vitro repair of 1meA and 3meC in double-stranded DNA by nuclear extracts depended primarily, if not solely, on mABH2. Our data suggest that mABH2 and mABH3 have different roles in the defense against alkylating agents.     

Empirical support for optimal virulence in a castrating parasite

The trade-off hypothesis for the evolution of virulence predicts that parasite transmission stage production and host exploitation are balanced such that lifetime transmission success (LTS) is maximised. However, the experimental evidence for this prediction is weak, mainly because LTS, which indicates parasite fitness, has been difficult to measure. For castrating parasites, this simple model has been modified to take into account that parasites convert host reproductive resources into transmission stages. Parasites that kill the host too early will hardly benefit from these resources, while postponing the killing of the host results in diminished returns. As predicted from optimality models, a parasite inducing castration should therefore castrate early, but show intermediate levels of virulence, where virulence is measured as time to host killing. We studied virulence in an experimental system where a bacterial parasite castrates its host and produces spores that are not released until after host death. This permits estimating the LTS of the parasite, which can then be related to its virulence. We exposed replicate individual Daphnia magna (Crustacea) of one host clone to the same amount of bacterial spores and followed individuals until their death. We found that the parasite shows strong variation in the time to kill its host and that transmission stage production peaks at an intermediate level of virulence. A further experiment tested for the genetic basis of variation in virulence by comparing survival curves of daphniids infected with parasite spores obtained from early killing versus late killing infections. Hosts infected with early killer spores had a significantly higher death rate as compared to those infected with late killers, indicating that variation in time to death was at least in part caused by genetic differences among parasites. We speculate that the clear peak in lifetime reproductive success at intermediate killing times may be caused by the exceptionally strong physiological trade-off between host and parasite reproduction. This is the first experimental study to demonstrate that the production of propagules is highest at intermediate levels of virulence and that parasite genetic variability is available to drive the evolution of virulence in this system.     

Synthetic Effects of secG and secY2 Mutations on Exoproteome Biogenesis in Staphylococcus aureus

The Gram-positive pathogen Staphylococcus aureus secretes various proteins into its extracellular milieu. Bioinformatics analyses have indicated that most of these proteins are directed to the canonical Sec pathway, which consists of the translocation motor SecA and a membrane-embedded channel composed of the SecY, SecE, and SecG proteins. In addition, S. aureus contains an accessory Sec2 pathway involving the SecA2 and SecY2 proteins. Here, we have addressed the roles of the nonessential channel components SecG and SecY2 in the biogenesis of the extracellular proteome of S. aureus. The results show that SecG is of major importance for protein secretion by S. aureus. Specifically, the extracellular accumulation of nine abundant exoproteins and seven cell wall-bound proteins was significantly affected in an secG mutant. No secretion defects were detected for strains with a secY2 single mutation. However, deletion of secY2 exacerbated the secretion defects of secG mutants, affecting the extracellular accumulation of one additional exoprotein and one cell wall protein. Furthermore, an secG secY2 double mutant displayed a synthetic growth defect. This might relate to a slightly elevated expression of sraP, encoding the only known substrate for the Sec2 pathway, in cells lacking SecG. Additionally, the results suggest that SecY2 can interact with the Sec1 channel, which would be consistent with the presence of a single set of secE and secG genes in S. aureus.Staphylococcus aureus is a well-represented component of the human microbiota as nasal carriage of this Gram-positive bacterium has been shown for 30 to 40% of the population (32). This organism can, however, turn into a dangerous pathogen that is able to infect almost every tissue in the human body. S. aureus has become particularly notorious for its high potential to develop resistance against commonly used antibiotics (20, 49). Accordingly, the S. aureus genome encodes an arsenal of virulence factors that can be expressed when needed at different stages of growth. These include surface proteins and invasins that are necessary for colonization of host tissues, surface-exposed factors for evasion of the immune system, exotoxins for the subversion of protective host barriers, and resistance proteins for protection against antimicrobial agents (37, 57).Most proteinaceous virulence factors of S. aureus are synthesized as precursors with an N-terminal signal peptide to direct their transport from the cytoplasm across the membrane to an extracytoplasmic location, such as the cell wall or the extracellular milieu (38, 45). As shown for various Gram-positive bacteria, the signal peptides of S. aureus are generally longer and more hydrophobic than those of Gram-negative bacteria (38, 54). On the basis of signal peptide predictions using a variety of algorithms, it is believed that most exoproteins of S. aureus are exported to extracytoplasmic locations via the general secretory (Sec) pathway (38). This seems to involve precursor targeting to the Sec machinery via the signal recognition particle instead of the well-characterized proteobacterial chaperone SecB, which is absent from Gram-positive bacteria (16, 19, 53). The preproteins are then bound by the translocation motor protein SecA (38, 45). Through repeated cycles of ATP binding and hydrolysis, SecA pushes the protein in an unfolded state through the membrane-embedded SecYEG translocation channel (12, 30, 33, 52). Upon initiation of the translocation process, the proton motive force is thought to accelerate preprotein translocation through the Sec channel (26). Recently, the structure of the SecA/SecYEG complex from the Gram-negative bacterium Thermotoga maritima was solved at 4.5 Å resolution (58). In this structure, one SecA molecule is bound to one set of SecYEG channel proteins. The core of the Sec translocon consists of the SecA, SecY, and SecE proteins, which are essential for growth and viability of bacteria, such as Escherichia coli and Bacillus subtilis (6, 9, 22). In contrast, the channel component SecG is dispensable for growth, cell viability, and protein translocation (26, 48). Nevertheless, SecG does enhance the efficiency of preprotein translocation through the SecYE channel (26, 48). This is of particular relevance at low temperatures and in the absence of a proton motive force (17). Several studies suggest that E. coli SecG undergoes topology inversion during preprotein translocation (25, 27, 43). Even so, van der Sluis et al. reported that SecG cross-linked to SecY is fully functional despite its fixed topology (46). During or shortly after membrane translocation of a preprotein through the Sec channel, the signal peptide is removed by signal peptidase. This is a prerequisite for the release of the translocated protein from the membrane (1, 47).Several pathogens, including Streptococcus gordonii, Streptococcus pneumoniae, Bacillus anthracis, Bacillus cereus, and S. aureus, contain a second set of chromosomal secA and secY genes named secA2 and secY2, respectively (39). Comparison of the amino acid sequences of the SecY1 and SecY2 proteins shows that their similarity is low (about 20% identity) and that the conserved regions are mainly restricted to the membrane-spanning domains. It has been shown for S. gordonii that the transport of at least one protein is dependent on the presence of SecA2 and SecY2. This protein, GspB, is a large cell surface glycoprotein that is involved in platelet binding (4). The protein contains an unusually long N-terminal signal peptide of 90 amino acids, large serine-rich repeats, and a C-terminal LPXTG motif for covalent cell wall binding. The gspB gene is located in a gene cluster with the secA2 and secY2 genes. Two other genes in this cluster encode the glycosylation proteins GftA and GftB, which seem to be necessary for stabilization of pre-GspB. Furthermore, the asp4 and asp5 genes in the secA2 secY2 gene cluster show similarity to secE and secG, and they are important for GspB export by S. gordonii (44). Despite this similarity, SecE and SecG cannot complement for the absence of Asp4 and Asp5, respectively. The secA2-secY2 gene cluster is also present in S. aureus, but homologues of the asp4 and asp5 genes are lacking. This seems to suggest that SecA2 and SecY2 of S. aureus share the SecE and SecG proteins with SecA1 and SecY1. The sraP gene in the secA2-secY2 gene cluster of S. aureus encodes a protein with features similar to those described for GspB. Siboo and colleagues (41) have shown that SraP is glycosylated and capable of binding to platelets. Importantly, the disruption of sraP resulted in a decreased ability to initiate infective endocarditis in a rabbit model. Consistent with the findings in S. gordonii, SraP export was shown to depend on SecA2/SecY2 (40). However, it has remained unclear whether other S. aureus proteins are also translocated across the membrane in an SecA2/SecY2-dependent manner.The present studies were aimed at defining the roles of two Sec channel components, SecG and SecY2, in the biogenesis of the S. aureus exoproteome. The results show that secG and secY2 are not essential for growth and viability of S. aureus. While the absence of SecY2 by itself had no detectable effect, the absence of SecG had a profound impact on the composition of the exoproteome of S. aureus. Various extracellular proteins were present in decreased amounts in the growth medium of secG mutant strains, which is consistent with impaired Sec channel function. However, a few proteins were present in increased amounts. Furthermore, the absence of secG caused a serious decrease in the amounts of the cell wall-bound Sbi protein. Most notable, a secG secY2 double mutant strain displayed synthetic growth and secretion defects.     

Latent heat exchange in the boreal and arctic biomes

In this study latent heat flux (λE) measurements made at 65 boreal and arctic eddy‐covariance (EC) sites were analyses by using the Penman–Monteith equation. Sites were stratified into nine different ecosystem types: harvested and burnt forest areas, pine forests, spruce or fir forests, Douglas‐fir forests, broadleaf deciduous forests, larch forests, wetlands, tundra and natural grasslands. The Penman–Monteith equation was calibrated with variable surface resistances against half‐hourly eddy‐covariance data and clear differences between ecosystem types were observed. Based on the modeled behavior of surface and aerodynamic resistances, surface resistance tightly control λE in most mature forests, while it had less importance in ecosystems having shorter vegetation like young or recently harvested forests, grasslands, wetlands and tundra. The parameters of the Penman–Monteith equation were clearly different for winter and summer conditions, indicating that phenological effects on surface resistance are important. We also compared the simulated λE of different ecosystem types under meteorological conditions at one site. Values of λE varied between 15% and 38% of the net radiation in the simulations with mean ecosystem parameters. In general, the simulations suggest that λE is higher from forested ecosystems than from grasslands, wetlands or tundra‐type ecosystems. Forests showed usually a tighter stomatal control of λE as indicated by a pronounced sensitivity of surface resistance to atmospheric vapor pressure deficit. Nevertheless, the surface resistance of forests was lower than for open vegetation types including wetlands. Tundra and wetlands had higher surface resistances, which were less sensitive to vapor pressure deficits. The results indicate that the variation in surface resistance within and between different vegetation types might play a significant role in energy exchange between terrestrial ecosystems and atmosphere. These results suggest the need to take into account vegetation type and phenology in energy exchange modeling.     

Explorative multivariate analyses of 16S rRNA gene data from microbial communities in modified-atmosphere-packed salmon and coalfish

Modified-atmosphere packaging (MAP) of foods in combination with low-temperature storage extends product shelf life by limiting microbial growth. We investigated the microbial biodiversity of MAP salmon and coalfish by using an explorative approach and analyzing both the total amounts of bacteria and the microbial group composition (both aerobic and anaerobic bacteria). Real-time PCR analyses revealed a surprisingly large difference in the microbial loads for the different fish samples. The microbial composition was determined by examining partial 16S rRNA gene sequences from 180 bacterial isolates, as well as by performing terminal restriction fragment length polymorphism analysis and cloning 92 sequences from PCR products of DNA directly retrieved from the fish matrix. Twenty different bacterial groups were identified. Partial least-squares (PLS) regression was used to relate the major groups of bacteria identified to the fish matrix and storage time. A strong association of coalfish with Photobacterium phosphoreum was observed. Brochothrix spp. and Carnobacterium spp., on the other hand, were associated with salmon. These bacteria dominated the fish matrixes after a storage period. Twelve Carnobacterium isolates were identified as either Carnobacterium piscicola (five isolates) or Carnobacterium divergens (seven isolates), while the eight Brochothrix isolates were identified as Brochothrix thermosphacta by full-length 16S rRNA gene sequencing. Principal-component analyses and PLS analysis of the growth characteristics (with 49 different substrates) showed that C. piscicola had distinct substrate requirements, while the requirements of B. thermosphacta and C. piscicola were quite divergent. In conclusion, our explorative multivariate approach gave a picture of the total microbial biodiversity in MAP fish that was more comprehensive than the picture that could be obtained previously. Such information is crucial in controlled food production when, for example, the hazard analysis of critical control points principle is used.     

Use of the novel Plk1 inhibitor ZK-thiazolidinone to elucidate functions of Plk1 in early and late stages of mitosis

Polo-like kinase 1 (Plk1) is a key regulator of mitotic progression and cell division in eukaryotes. It is highly expressed in tumor cells and considered a potential target for cancer therapy. Here, we report the discovery and application of a novel potent small-molecule inhibitor of mammalian Plk1, ZK-Thiazolidinone (TAL). We have extensively characterized TAL in vitro and addressed TAL specificity within cells by studying Plk1 functions in sister chromatid separation, centrosome maturation, and spindle assembly. Moreover, we have used TAL for a detailed analysis of Plk1 in relation to PICH and PRC1, two prominent interaction partners implicated in spindle assembly checkpoint function and cytokinesis, respectively. Specifically, we show that Plk1, when inactivated by TAL, spreads over the arms of chromosomes, resembling the localization of its binding partner PICH, and that both proteins are mutually dependent on each other for correct localization. Finally, we show that Plk1 activity is essential for cleavage furrow formation and ingression, leading to successful cytokinesis.     

APOMIXIS AND POLYEMBRYONY IN HIEROCHLOË ODORATA

Norstog , Knut . (Wittenberg U., Springfield, Ohio.) Apomixis and polyembryony in Hierochloe odorata. Amer. Jour. Bot. 50(8): 815–821. Illus. 1963.—Hierochloë odorata from Michigan, having 2n = 56 chromosomes, was found to reproduce by pseudogamous fertilization of diploid aposporous embryo sacs. Diploid embryos, 2n= 56, and 5n = 140 ± endosperm occurred together. Megasporogenesis was incomplete, and aposporous embryo sac initials developed directly into 8-nucleate Polygonum type embryo sacs. Microsporogenesis was irregular with univalent, bivalent and multivalent chromosomes in meiosis-I. Dyads and microspores varied between n = 24 and n = 32, and less than 50% of the pollen grains stained with acetocarmine. Two other races of H. odorata are known to occur in North America. They are an apparently infertile type in Canada with 2n = 28, and a perfectly fertile race from coastal Connecticut also with 2n = 28. It is suggested that the H. odorata with 2n = 56 is a derivative of the sterile Canadian race.