Sven Klages

Genome Expansion and Gene Loss in Powdery Mildew Fungi Reveal Tradeoffs in Extreme Parasitism

From Blight to Powdery Mildew Pathogenic effects of microbes on plants have widespread consequences. Witness, for example, the cultural upheavals driven by potato blight in the 1800s. A variety of microbial pathogens continue to afflict crop plants today, driving both loss of yield and incurring the increased costs of control mechanisms. Now, four reports analyze microbial genomes in order to understand better how plant pathogens function (see the Perspective by Dodds). Raffaele et al. (p. 1540) describe how the genome of the potato blight pathogen accommodates transfer to different hosts. Spanu et al. (p. 1543) analyze what it takes to be an obligate biotroph in barley powdery mildew, and Baxter et al. (p. 1549) ask a similar question for a natural pathogen of Arabidopsis. Schirawski et al. (p. 1546) compared genomes of maize pathogens to identify virulence determinants. Better knowledge of what in a genome makes a pathogen efficient and deadly is likely to be useful for improving agricultural crop management and breeding. A group of papers analyzes pathogen genomes to find the roots of virulence, opportunism, and life-style determinants. Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248 candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a core set conserved in all three mildews, suggesting that most effectors represent species-specific adaptations.

Nephrocystin-5, a ciliary IQ domain protein, is mutated in Senior-Loken syndrome and interacts with RPGR and calmodulin

Nephronophthisis (NPHP) is the most frequent genetic cause of chronic renal failure in children. Identification of four genes mutated in NPHP subtypes 1–4 (refs. 4–9) has linked the pathogenesis of NPHP to ciliary functions. Ten percent of affected individuals have retinitis pigmentosa, constituting the renal-retinal Senior-Loken syndrome (SLSN). Here we identify, by positional cloning, mutations in an evolutionarily conserved gene, IQCB1 (also called NPHP5), as the most frequent cause of SLSN. IQCB1 encodes an IQ-domain protein, nephrocystin-5. All individuals with IQCB1 mutations have retinitis pigmentosa. Hence, we examined the interaction of nephrocystin-5 with RPGR (retinitis pigmentosa GTPase regulator), which is expressed in photoreceptor cilia and associated with 10–20% of retinitis pigmentosa. We show that nephrocystin-5, RPGR and calmodulin can be coimmunoprecipitated from retinal extracts, and that these proteins localize to connecting cilia of photoreceptors and to primary cilia of renal epithelial cells. Our studies emphasize the central role of ciliary dysfunction in the pathogenesis of SLSN.

SNP and haplotype mapping for genetic analysis in the rat.

The laboratory rat is one of the most extensively studied model organisms. Inbred laboratory rat strains originated from limited Rattus norvegicus founder populations, and the inherited genetic variation provides an excellent resource for the correlation of genotype to phenotype. Here, we report a survey of genetic variation based on almost 3 million newly identified SNPs. We obtained accurate and complete genotypes for a subset of 20,238 SNPs across 167 distinct inbred rat strains, two rat recombinant inbred panels and an F2 intercross. Using 81% of these SNPs, we constructed high-density genetic maps, creating a large dataset of fully characterized SNPs for disease gene mapping. Our data characterize the population structure and illustrate the degree of linkage disequilibrium. We provide a detailed SNP map and demonstrate its utility for mapping of quantitative trait loci. This community resource is openly available and augments the genetic tools for this workhorse of physiological studies.

European sea bass genome and its variation provide insights into adaptation to euryhalinity and speciation

The European sea bass (Dicentrarchus labrax) is a temperate-zone euryhaline teleost of prime importance for aquaculture and fisheries. This species is subdivided into two naturally hybridizing lineages, one inhabiting the north-eastern Atlantic Ocean and the other the Mediterranean and Black seas. Here we provide a high-quality chromosome-scale assembly of its genome that shows a high degree of synteny with the more highly derived teleosts. We find expansions of gene families specifically associated with ion and water regulation, highlighting adaptation to variation in salinity. We further generate a genome-wide variation map through RAD-sequencing of Atlantic and Mediterranean populations. We show that variation in local recombination rates strongly influences the genomic landscape of diversity within and differentiation between lineages. Comparing predictions of alternative demographic models to the joint allele-frequency spectrum indicates that genomic islands of differentiation between sea bass lineages were generated by varying rates of introgression across the genome following a period of geographical isolation.

Genomic insights into the physiology and ecology of the marine filamentous cyanobacterium Lyngbya majuscula.

Filamentous cyanobacteria of the genus Lyngbya are important contributors to coral reef ecosystems, occasionally forming dominant cover and impacting the health of many other co-occurring organisms. Moreover, they are extraordinarily rich sources of bioactive secondary metabolites, with 35% of all reported cyanobacterial natural products deriving from this single pantropical genus. However, the true natural product potential and life strategies of Lyngbya strains are poorly understood because of phylogenetic ambiguity, lack of genomic information, and their close associations with heterotrophic bacteria and other cyanobacteria. To gauge the natural product potential of Lyngbya and gain insights into potential microbial interactions, we sequenced the genome of Lyngbya majuscula 3L, a Caribbean strain that produces the tubulin polymerization inhibitor curacin A and the molluscicide barbamide, using a combination of Sanger and 454 sequencing approaches. Whereas ∼293,000 nucleotides of the draft genome are putatively dedicated to secondary metabolism, this is far too few to encode a large suite of Lyngbya metabolites, suggesting Lyngbya metabolites are strain specific and may be useful in species delineation. Our analysis revealed a complex gene regulatory network, including a large number of sigma factors and other regulatory proteins, indicating an enhanced ability for environmental adaptation or microbial associations. Although Lyngbya species are reported to fix nitrogen, nitrogenase genes were not found in the genome or by PCR of genomic DNA. Subsequent growth experiments confirmed that L. majuscula 3L is unable to fix atmospheric nitrogen. These unanticipated life history characteristics challenge current views of the genus Lyngbya.

A 454 sequencing approach for large scale phylogenomic analysis of the common emperor scorpion (Pandinus imperator).

In recent years, phylogenetic tree reconstructions that rely on multiple gene alignments that had been deduced from expressed sequence tags (ESTs) have become a popular method in molecular systematics. Here, we present a 454 pyrosequencing approach to infer the transcriptome of the Emperor scorpion Pandinus imperator. We obtained 428,844 high-quality reads (mean length=223+/-50 b) from total cDNA, which were assembled into 8334 contigs (mean length 422+/-313 bp) and 26,147 singletons. About 1200 contigs were successfully annotated by BLAST and orthology search. Specific analyses of eight distinct hemocyanin sequences provided further proof for the quality of the 454 reads and the assembly process. The P. imperator sequences were included in a concatenated alignment of 149 orthologous genes of 67 metazoan taxa that covers 39,842 amino acids. After removal of low-quality regions, 11,168 positions were employed for phylogenetic reconstructions. Using Bayesian and maximum likelihood methods, we obtained strongly supported monophyletic Ecdysozoa, Arthropoda (excluding Tardigrada), Euarthropoda, Pancrustacea and Hexapoda. We also recovered the Myriochelata (Chelicerata+Myriapoda). Within the chelicerates, Pycnogonida form the sister group of Euchelicerata. However, Arachnida were found paraphyletic because the Acari (mites and ticks) were recovered as sister group of a clade comprising Xiphosura, Scorpiones and Araneae. In summary, we have shown that 454 pyrosequencing is a cost-effective method that provides sufficient data and coverage depth for gene detection and multigene-based phylogenetic analyses.

Comparative Genomic Analysis of Fruiting Body Formation in Myxococcales

Genetic programs underlying multicellular morphogenesis and cellular differentiation are most often associated with eukaryotic organisms, but examples also exist in bacteria such as the formation of multicellular, spore-filled fruiting bodies in the order Myxococcales. Most members of the Myxococcales undergo a multicellular developmental program culminating in the formation of spore-filled fruiting bodies in response to starvation. To gain insight into the evolutionary history of fruiting body formation in Myxococcales, we performed a comparative analysis of the genomes and transcriptomes of five Myxococcales species, four of these undergo fruiting body formation (Myxococcus xanthus, Stigmatella aurantiaca, Sorangium cellulosum, and Haliangium ochraceum) and one does not (Anaeromyxobacter dehalogenans). Our analyses show that a set of 95 known M. xanthus development-specific genes--although suffering from a sampling bias--are overrepresented and occur more frequently than an average M. xanthus gene in S. aurantiaca, whereas they occur at the same frequency as an average M. xanthus gene in S. cellulosum and in H. ochraceum and are underrepresented in A. dehalogenans. Moreover, genes for entire signal transduction pathways important for fruiting body formation in M. xanthus are conserved in S. aurantiaca, whereas only a minority of these genes are conserved in A. dehalogenans, S. cellulosum, and H. ochraceum. Likewise, global gene expression profiling of developmentally regulated genes showed that genes that upregulated during development in M. xanthus are overrepresented in S. aurantiaca and slightly underrepresented in A. dehalogenans, S. cellulosum, and H. ochraceum. These comparative analyses strongly indicate that the genetic programs for fruiting body formation in M. xanthus and S. aurantiaca are highly similar and significantly different from the genetic program directing fruiting body formation in S. cellulosum and H. ochraceum. Thus, our analyses reveal an unexpected level of plasticity in the genetic programs for fruiting body formation in the Myxococcales and strongly suggest that the genetic program underlying fruiting body formation in different Myxococcales is not conserved. The evolutionary implications of this finding are discussed.

Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica.

Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis—the paradigm of mesophilic hydrocarbonoclastic bacteria—O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.

Comparative analysis of expressed sequence tag (EST) libraries in the seagrass Zostera marina subjected to temperature stress

Global warming is associated with increasing stress and mortality on temperate seagrass beds, in particular during periods of high sea surface temperatures during summer months, adding to existing anthropogenic impacts, such as eutrophication and habitat destruction. We compare several expressed sequence tag (EST) in the ecologically important seagrass Zostera marina (eelgrass) to elucidate the molecular genetic basis of adaptation to environmental extremes. We compared the tentative unigene (TUG) frequencies of libraries derived from leaf and meristematic tissue from a control situation with two experimentally imposed temperature stress conditions and found that TUG composition is markedly different among these conditions (all P < 0.0001). Under heat stress, we find that 63 TUGs are differentially expressed (d.e.) at 25°C compared with lower, no-stress condition temperatures (4°C and 17°C). Approximately one-third of d.e. eelgrass genes were characteristic for the stress response of the terrestrial plant model Arabidopsis thaliana. The changes in gene expression suggest complex photosynthetic adjustments among light-harvesting complexes, reaction center subunits of photosystem I and II, and components of the dark reaction. Heat shock encoding proteins and reactive oxygen scavengers also were identified, but their overall frequency was too low to perform statistical tests. In all conditions, the most abundant transcript (3–15%) was a putative metallothionein gene with unknown function. We also find evidence that heat stress may translate to enhanced infection by protists. A total of 210 TUGs contain one or more microsatellites as potential candidates for gene-linked genetic markers. Data are publicly available in a user-friendly database at http://www.uni-muenster.de/Evolution/ebb/Services/zostera.

Genome of Streptococcus oralis strain Uo5.

Streptococcus oralis, a commensal species of the human oral cavity, belongs to the Mitis group of streptococci, which includes one of the major human pathogens as well, S. pneumoniae. We report here the first complete genome sequence of this species. S. oralis Uo5, a high-level penicillin- and multiple-antibiotic-resistant isolate from Hungary, is competent for genetic transformation under laboratory conditions. Comparative and functional genomics of Uo5 will be important in understanding the evolution of pathogenesis among Mitis streptococci and their potential to engage in interspecies gene transfer.