Cyanobacteria and the Great Oxidation Event: evidence from genes and fossils

Cyanobacteria are among the most ancient of evolutionary lineages, oxygenic photosynthesizers that may have originated before 3.0 Ga, as evidenced by free oxygen levels. Throughout the Precambrian, cyanobacteria were one of the most important drivers of biological innovations, strongly impacting early Earth's environments. At the end of the Archean Eon, they were responsible for the rapid oxygenation of Earth's atmosphere during an episode referred to as the Great Oxidation Event (GOE). However, little is known about the origin and diversity of early cyanobacterial taxa, due to: (1) the scarceness of Precambrian fossil deposits; (2) limited characteristics for the identification of taxa; and (3) the poor preservation of ancient microfossils. Previous studies based on 16S rRNA have suggested that the origin of multicellularity within cyanobacteria might have been associated with the GOE. However, single‐gene analyses have limitations, particularly for deep branches. We reconstructed the evolutionary history of cyanobacteria using genome scale data and re‐evaluated the Precambrian fossil record to get more precise calibrations for a relaxed clock analysis. For the phylogenomic reconstructions, we identified 756 conserved gene sequences in 65 cyanobacterial taxa, of which eight genomes have been sequenced in this study. Character state reconstructions based on maximum likelihood and Bayesian phylogenetic inference confirm previous findings, of an ancient multicellular cyanobacterial lineage ancestral to the majority of modern cyanobacteria. Relaxed clock analyses provide firm support for an origin of cyanobacteria in the Archean and a transition to multicellularity before the GOE. It is likely that multicellularity had a greater impact on cyanobacterial fitness and thus abundance, than previously assumed. Multicellularity, as a major evolutionary innovation, forming a novel unit for selection to act upon, may have served to overcome evolutionary constraints and enabled diversification of the variety of morphotypes seen in cyanobacteria today.     

Diversity and evolutionary patterns of immune genes in free-ranging Namibian leopards (Panthera pardus pardus)

The genes of the major histocompatibility complex (MHC) are a key component of the mammalian immune system and have become important molecular markers for fitness-related genetic variation in wildlife populations. Currently, no information about the MHC sequence variation and constitution in African leopards exists. In this study, we isolated and characterized genetic variation at the adaptively most important region of MHC class I and MHC class II-DRB genes in 25 free-ranging African leopards from Namibia and investigated the mechanisms that generate and maintain MHC polymorphism in the species. Using single-stranded conformation polymorphism analysis and direct sequencing, we detected 6 MHC class I and 6 MHC class II-DRB sequences, which likely correspond to at least 3 MHC class I and 3 MHC class II-DRB loci. Amino acid sequence variation in both MHC classes was higher or similar in comparison to other reported felids. We found signatures of positive selection shaping the diversity of MHC class I and MHC class II-DRB loci during the evolutionary history of the species. A comparison of MHC class I and MHC class II-DRB sequences of the leopard to those of other felids revealed a trans-species mode of evolution. In addition, the evolutionary relationships of MHC class II-DRB sequences between African and Asian leopard subspecies are discussed.     

Surface characteristics of grasslands in Inner Mongolia as detected by micrometeorological measurements

A roving tower concept was used to compare a semi-arid grassland site in Inner Mongolia (China), which was fenced in 1979 and ungrazed thereafter (UG79) with differently grazed semi-arid steppe ecosystems. The study was conducted during three consecutive years characterised by contrasting precipitation. The different grazing intensities included continuously and moderately grazed (CG), winter grazed (WG), and heavily grazed (HG). Here, we compare the energy fluxes and surface parameters that characterise the differently managed plots. The main focus is on sensible heat flux (H), available energy (AE), surface temperature (T ( s )), and surface albedo (alpha). Systematic errors were excluded by a side-to-side intercomparison of the instruments, and systematic climatic differences were minimised by the close distance between the fixed and the roving eddy covariance tower. Statistically, AE and T ( s ) were always significantly different between two simultaneously measured grazing intensities. Whereas AE was higher at UG79 in all years (mean difference of about 19Wm(-2)), T ( s ) was typically lower at UG79 (mean differences of 0.4 degrees C to about 2 degrees C). The exception was the end of the vegetation period in 2004 when T ( s ) was 0.6 degrees C higher at UG79 compared to CG. At UG79 alpha was typically significantly lower, and H was typically significantly higher. Consequently, latent heat fluxes (both as energy balance residual and directly measured) do not differ much between the different grazing intensities. It is concluded, that (1) the roving tower concept is able to detect differences due to grazing, (2) differences between the sites can be attributed to real surface differences, and (3) differences due to grazing intensities are small compared to interannual differences in surface fluxes.     

Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum

A series of experiments reported in the literature using fluxomics as an efficient functional genomics tool revealed that the L-lysine production of the Corynebacterium glutamicum strain MH20-22B correlates with the extent of intracellular NADPH supply. Some alternative metabolic engineering strategies to increase intracellular NADPH supply in the C. glutamicum strain DSM5715 were considered and finally the redirection of carbon flux through the pentose phosphate pathway with two NADPH generating enzymatic reactions was favored. Elsewhere, the construction of a phosphoglucose isomerase (Pgi) null mutant of the C. glutamicum strain DSM5715 has been described by utilizing genetic engineering as well as some aspects of its metabolic phenotype. Most interestingly, it was shown that not only could the L-lysine formation be increased by 1.7-fold but the by-product concentration for the null mutant strain was also able to be drastically reduced. In this publication we discuss this metabolic phenotype in detail and present additional data on by-product formation as well as yield considerations. Results from isotope based metabolic flux analysis in combination with considerations on NADPH metabolism clearly exclude the existence of Pgi isoenzymes in C. glutamicum strain DSM5715. The genome region containing the pgi gene was analyzed. It cannot be excluded that polar effects might have been caused by the disruption of the pgi gene and might have contributed to the observed metabolic phenotype of C. glutamicum Pgi mutants. We illustrate growth characteristics of a Pgi mutant of an industrial L-lysine production strain. A reduced growth rate and a biphasic growth behavior was observed. The importance of NADPH reoxidation for well balanced growth in Pgi mutants is discussed. Another phosphoglucose isomerase mutant of C. glutamicum has been described in literature with which an increase in L-lysine yield from 42 to 52% was observed. This finding highlights the general potential of metabolic flux redirection towards the pentose phosphate pathway, which could be used for metabolic engineering of the biotechnological synthesis of (1) aromatic amino acids and (2) chemicals whose synthesis depends on intracellular NADPH supply.     

A cyclophilin-like peptidyl-prolyl cis/trans isomerase from Legionella pneumophila – characterization, molecular cloning and overexpression

Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires’disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPIases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPiase at a level of enzyme activity comparable to that of FKBP25mem. The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPIases. The Icy gene (Legionella cycophn) was cloned and sequenced. It encodes a putative 164-amino-acid protein with a molecular mass of 17 968 Da called L. pneumophila cyclophilin 18 (L. p. Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coll with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histi-dine tag and an enterokinase cleavage site exhibits     

The apparent uptake of fluorescently labeled siRNAs by electroporated cells depends on the fluorochrome

Transfection of mammalian cells with preformed small interfering RNAs (siRNAs) permits a transient and often specific reduction of gene expression. It is possible to rapidly examine the uptake of siRNAs by transfection with fluorescently labeled siRNAs. We examined the apparent uptake of such siRNAs by several leukemic cell lines after electroporation. We show that Cy3 and Cy5-labeled siRNAs cause a significant amount of cell fluorescence, as judged by flow cytometry. In contrast, several fluorescein-labeled siRNAs could not be detected. Nevertheless, such fluoresceinated siRNAs efficiently suppressed a leukemic target gene, demonstrating that siRNA uptake must have taken place. Therefore, for cell electroporation, fluorescein-labeled siRNAs may lead to false negative results and should not be used to examine electroporation-mediated siRNA uptake.     

Arbuscular mycorrhizal colonization of halophytes in Central European salt marshes

 Halophytes from both coastal and inland Central European salt marshes were examined for colonization by arbuscular mycorrhizal (AM) fungi. Plants from different families were strongly colonized but the degree of colonization varied with the individual plant and apparently during the vegetation period, too. Members of the typical non-mycorrhizal families like Armeria maritima of the Plumbaginaceae and Salicornia europaea of the Chenopodiaceae were found to be colonized, particularly in the drier salt marshes. High numbers of Glomus spores were found in the saline soils, especially those of the inland locations examined. Approximately 80% of these spores were from Glomus geosporum as shown by a typical restriction fragment length polymorphism (RFLP) pattern of the amplified internal transcribed spacer regions. The present study demonstrates that RFLP analysis is useful when screening habitats for the occurrence of mycorrhizal fungi which can be identified only with difficulty by morphological criteria. Accepted: 25 August 2000     

M line-deficient titin causes cardiac lethality through impaired maturation of the sarcomere

Titin, the largest protein known to date, has been linked to sarcomere assembly and function through its elastic adaptor and signaling domains. Titin's M-line region contains a unique kinase domain that has been proposed to regulate sarcomere assembly via its substrate titin cap (T-cap). In this study, we use a titin M line-deficient mouse to show that the initial assembly of the sarcomere does not depend on titin's M-line region or the phosphorylation of T-cap by the titin kinase. Rather, titin's M-line region is required to form a continuous titin filament and to provide mechanical stability of the embryonic sarcomere. Even without titin integrating into the M band, sarcomeres show proper spacing and alignment of Z discs and M bands but fail to grow laterally and ultimately disassemble. The comparison of disassembly in the developing and mature knockout sarcomere suggests diverse functions for titin's M line in embryonic development and the adult heart that not only involve the differential expression of titin isoforms but also of titin-binding proteins.     

Identification of equine P-selectin glycoprotein ligand-1 (CD162)

P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a dimeric, mucin-like, transmembrane glycoprotein constitutively expressed on leukocytes. A high baseline level of P-selectin expression in circulating equine platelets suggests a primed state toward inflammation and thrombosis via P-selectin/PSGL-1 adhesion. To investigate the potential role of equine P-selectin in these events, we first identified the cDNA sequence of equine PSGL-1 (ePSGL-1) using degenerate PCR and RACE-PCR and then compared the predicted sequence with that of human PSGL-1 (hPSGL-1). ePSGL-1 protein subunit is predicted to be 43 kDa and composed of 420 amino acids with a predicted 18-amino-acid signal sequence showing 78% homology to hPSGL-1. Previously published work has shown that binding of P-selectin requires sulfation of at least one of three tyrosines and O-glycosylation of one threonine in the N-terminus of human PSGL-1. However, the corresponding domain in ePSGL-1, spanning residues 19–43, contains only one tyrosine in the vicinity of two threonines at positions 25 and 41. ePSGL-1 contains 14 threonine/serine-rich decameric repeats as compared to hPSGL-1 which contains 14–16 threonine-rich decameric repeats. The transmembrane and cytoplasmic domains display 91% and 74% homology to corresponding human PSGL-1 domains, respectively. In summary, there is 71% homology in comparing the open reading frame (ORF) of ePSGL-1 with that of hPSGL-1. The greatest homologies between species exist in the transmembrane domain and cytoplasmic tail while substantial differences exist in the extracellular domain.The nucleotide sequence data reported in this article has been submitted to GenBank and assigned the accession number AY298766.