Publications by authors named "Andreas Brachmann"

58 Publications

Genome-Wide Identification of Essential and Auxiliary Gene Sets for Magnetosome Biosynthesis in Magnetospirillum gryphiswaldense.

mSystems 2020 Nov 17;5(6). Epub 2020 Nov 17.

Department of Microbiology, University of Bayreuth, Bayreuth, Germany

Magnetotactic bacteria (MTB) stand out by their ability to manufacture membrane-enclosed magnetic organelles, so-called magnetosomes. Previously, it has been assumed that a genomic region of approximately 100 kbp, the magnetosome island (MAI), harbors all genetic determinants required for this intricate biosynthesis process. Recent evidence, however, argues for the involvement of additional auxiliary genes that have not been identified yet. In the present study, we set out to delineate the full gene complement required for magnetosome production in the alphaproteobacterium using a systematic genome-wide transposon mutagenesis approach. By an optimized procedure, a Tn insertion library of 80,000 clones was generated and screened, yielding close to 200 insertants with mild to severe impairment of magnetosome biosynthesis. Approximately 50% of all Tn insertion sites mapped within the MAI, mostly leading to a nonmagnetic phenotype. In contrast, in the majority of weakly magnetic Tn insertion mutants, genes outside the MAI were affected, which typically caused lower numbers of magnetite crystals with partly aberrant morphology, occasionally combined with deviant intracellular localization. While some of the Tn-struck genes outside the MAI belong to pathways that have been linked to magnetosome formation before (e.g., aerobic and anaerobic respiration), the majority of affected genes are involved in so far unsuspected cellular processes, such as sulfate assimilation, oxidative protein folding, and cytochrome maturation, or are altogether of unknown function. We also found that signal transduction and redox functions are enriched in the set of Tn hits outside the MAI, suggesting that such processes are particularly important in support of magnetosome biosynthesis. is one of the few tractable model magnetotactic bacteria (MTB) for studying magnetosome biomineralization. So far, knowledge on the genetic determinants of this complex process has been mainly gathered using reverse genetics and candidate approaches. In contrast, nontargeted forward genetics studies are lacking, since application of such techniques in MTB has been complicated for a number of technical reasons. Here, we report on the first comprehensive transposon mutagenesis study in MTB, aiming at systematic identification of auxiliary genes necessary to support magnetosome formation in addition to key genes harbored in the magnetosome island (MAI). Our work considerably extends the candidate set of novel subsidiary determinants and shows that the full gene complement underlying magnetosome biosynthesis is larger than assumed. In particular, we were able to define certain cellular pathways as specifically important for magnetosome formation that have not been implicated in this process so far.
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http://dx.doi.org/10.1128/mSystems.00565-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7676999PMC
November 2020

Involvement of MexS and MexEF-OprN in Resistance to Toxic Ion Chelators in KT2440.

Microorganisms 2020 Nov 14;8(11). Epub 2020 Nov 14.

Biozentrum, Mikrobiologie, Ludwig-Maximilians-Universität München, 82152 Martinsried, Germany.

Bacteria must be able to cope with harsh environments to survive. In Gram-negative bacteria like species, resistance-nodulation-division (RND) transporters contribute to this task by pumping toxic compounds out of cells. Previously, we found that the RND system TtgABC of KT2440 confers resistance to toxic metal chelators of the bipyridyl group. Here, we report that the incubation of a mutant in medium containing 2,2'-bipyridyl generated revertant strains able to grow in the presence of this compound. This trait was related to alterations in the pp_2827 locus (homolog of in ). The deletion and complementation of pp_2827 confirmed the importance of the locus for the revertant phenotype. Furthermore, alteration in the pp_2827 locus stimulated expression of the operon encoding an RND efflux pump. Deletion and complementation of confirmed that the latter system can compensate the growth defect of the mutant in the presence of 2,2'-bipyridyl. To our knowledge, this is the first report on a role of pp_2827 () in the regulation of in KT2440. The results expand the information about the significance of MexEF-OprN in the stress response of KT2440 and the mechanisms for coping with bipyridyl toxicity.
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http://dx.doi.org/10.3390/microorganisms8111782DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697342PMC
November 2020

Candidatus Frankia nodulisporulans sp. nov., an Alnus glutinosa-infective Frankia species unable to grow in pure culture and able to sporulate in-planta.

Syst Appl Microbiol 2020 Nov 28;43(6):126134. Epub 2020 Sep 28.

Department of Plant Physiology, UPSC, Umeå University, 90187 Umeå, Sweden. Electronic address:

We describe a new Frankia species, for three non-isolated strains obtained from Alnus glutinosa in France and Sweden, respectively. These strains can nodulate several Alnus species (A. glutinosa, A. incana, A. alnobetula), they form hyphae, vesicles and sporangia in the root nodule cortex but have resisted all attempts at isolation in pure culture. Their genomes have been sequenced, they are significantly smaller than those of other Alnus-infective species (5Mb instead of 7.5Mb) and are very closely related to one another (ANI of 100%). The name Candidatus Frankia nodulisporulans is proposed. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and draft genome sequences reported in this study for AgTrS, AgUmASt1 and AgUmASH1 are MT023539/LR778176/LR778180 and NZ_CADCWS000000000.1/CADDZU010000001/CADDZW010000001, respectively.
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http://dx.doi.org/10.1016/j.syapm.2020.126134DOI Listing
November 2020

Meiotic recombination in the offspring of Microbotryum hybrids and its impact on pathogenicity.

BMC Evol Biol 2020 09 17;20(1):123. Epub 2020 Sep 17.

AG Geobotanik, Ruhr-Universität Bochum, Universitätsstraße 150, 44780, Bochum, Germany.

Background: Hybridization is a central mechanism in evolution, producing new species or introducing important genetic variation into existing species. In plant-pathogenic fungi, adaptation and specialization to exploit a host species are key determinants of evolutionary success. Here, we performed experimental crosses between the two pathogenic Microbotryum species, M. lychnidis-dioicae and M. silenes-acaulis that are specialized to different hosts. The resulting offspring were analyzed on phenotypic and genomic levels to describe genomic characteristics of hybrid offspring and genetic factors likely involved in host-specialization.

Results: Genomic analyses of interspecific fungal hybrids revealed that individuals were most viable if the majority of loci were inherited from one species. Interestingly, species-specific loci were strictly controlled by the species' origin of the mating type locus. Moreover we detected signs of crossing over and chromosome duplications in the genomes of the analyzed hybrids. In Microbotryum, mitochondrial DNA was found to be uniparentally inherited from the a mating type. Genome comparison revealed that most gene families are shared and the majority of genes are conserved between the two species, indicating very similar biological features, including infection and pathogenicity processes. Moreover, we detected 211 candidate genes that were retained under host-driven selection of backcrossed lines. These genes and might therefore either play a crucial role in host specialization or be linked to genes that are essential for specialization.

Conclusion: The combination of genome analyses with experimental selection and hybridization is a promising way to investigate host-pathogen interactions. This study manifests genetic factors of host specialization that are required for successful biotrophic infection of the post-zygotic stage, but also demonstrates the strong influence of intra-genomic conflicts or instabilities on the viability of hybrids in the haploid host-independent stage.
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http://dx.doi.org/10.1186/s12862-020-01689-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499883PMC
September 2020

sp. nov., isolated from growing in the northern region of Tunisia.

Int J Syst Evol Microbiol 2020 Oct 8;70(10):5539-5550. Epub 2020 Sep 8.

Chair of Genetics, Faculty of Biology, Ludwig Maximilians University Munich, Germany.

Strain aSej3 was isolated from a root nodule of a plant growing in Bizerte, Tunisia. 16S rRNA gene analysis placed this strain within the genus . Multilocus sequence analysis (MLSA) including three housekeeping genes (, and ) grouped aSej3 together with CTAW71, CTAW11, RITF806, USDA 3051 and BTA-1. MLSA with five housekeeping genes (, , , and ) revealed that this strain shares less than 93.5 % nucleotide identity with other type strains. Genome sequencing and inspection revealed a genome size of 8.83 Mbp with a G+C content of 62.8 mol%. Genome-wide average nucleotide identity and digital DNA-DNA hybridization values were below 87.5 and 36.2 %, respectively, when compared to described species. Strain aSej3 nodulated plants under axenic conditions and its gene clustered within the genistearum symbiovar. Altogether, the phylogenetic data and the chemotaxonomic characteristics of this strain support that aSej3 represents a new species for which we propose the name sp. nov. with the type strain aSej3 (=DSM 108913=LMG 31020).
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http://dx.doi.org/10.1099/ijsem.0.004445DOI Listing
October 2020

More Filtering on SNP Calling Does Not Remove Evidence of Inter-Nucleus Recombination in Dikaryotic Arbuscular Mycorrhizal Fungi.

Front Plant Sci 2020 7;11:912. Epub 2020 Jul 7.

Department of Biology, University of Ottawa, Ottawa, ON, Canada.

Evidence for the existence of dikaryote-like strains, low nuclear sequence diversity and inter-nuclear recombination in arbuscular mycorrhizal fungi has been recently reported based on single nucleus sequencing data. Here, we aimed to support evidence of inter-nuclear recombination using an approach that filters SNP calls more conservatively, keeping only positions that are exclusively single copy and homozygous, and with at least five reads supporting a given SNP. This methodology recovers hundreds of putative inter-nucleus recombination events across publicly available sequence data from individual nuclei. Challenges related to the acquisition and analysis of sequence data from individual nuclei are highlighted and discussed, and ways to address these issues in future studies are presented.
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http://dx.doi.org/10.3389/fpls.2020.00912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7358544PMC
July 2020

A Novel Factor Essential for Unconventional Secretion of Chitinase Cts1.

Front Microbiol 2020 7;11:1529. Epub 2020 Jul 7.

Institute for Microbiology, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.

Subcellular targeting of proteins is essential to orchestrate cytokinesis in eukaryotic cells. During cell division of , for example, chitinases must be specifically targeted to the fragmentation zone at the site of cell division to degrade remnant chitin and thus separate mother and daughter cells. Chitinase Cts1 is exported to this location via an unconventional secretion pathway putatively operating in a lock-type manner. The underlying mechanism is largely unexplored. Here, we applied a forward genetic screen based on UV mutagenesis to identify components essential for Cts1 export. The screen revealed a novel factor termed Jps1 lacking known protein domains. Deletion of the corresponding gene confirmed its essential role for Cts1 secretion. Localization studies demonstrated that Jps1 colocalizes with Cts1 in the fragmentation zone of dividing yeast cells. While loss of Jps1 leads to exclusion of Cts1 from the fragmentation zone and strongly reduced unconventional secretion, deletion of the chitinase does not disturb Jps1 localization. Yeast-two hybrid experiments indicate that the two proteins might interact. In essence, we identified a novel component of unconventional secretion that functions in the fragmentation zone to enable export of Cts1. We hypothesize that Jps1 acts as an anchoring factor for Cts1.
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http://dx.doi.org/10.3389/fmicb.2020.01529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7358432PMC
July 2020

Genetic Underpinnings of Host Manipulation by as Revealed by Comparative Transcriptomics.

G3 (Bethesda) 2020 07 7;10(7):2275-2296. Epub 2020 Jul 7.

Department of Biology, College of Sciences, University of Central Florida, Orlando, Florida, 32816,

Ant-infecting fungi are globally distributed, host manipulating, specialist parasites that drive aberrant behaviors in infected ants, at a lethal cost to the host. An apparent increase in activity and wandering behaviors precedes a final summiting and biting behavior onto vegetation, which positions the manipulated ant in a site beneficial for fungal growth and transmission. We investigated the genetic underpinnings of host manipulation by: ( producing a high-quality hybrid assembly and annotation of the genome, () conducting laboratory infections coupled with RNAseq of and its host, , and () comparing these data to RNAseq data of and as a powerful method to identify gene expression patterns that suggest shared behavioral manipulation mechanisms across -ant species interactions. We propose differentially expressed genes tied to ant neurobiology, odor response, circadian rhythms, and foraging behavior may result by activity of putative fungal effectors such as enterotoxins, aflatrem, and mechanisms disrupting feeding behaviors in the ant.
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http://dx.doi.org/10.1534/g3.120.401290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341126PMC
July 2020

Draft Genome Sequence of mTS5, Isolated from in Tunisia.

Microbiol Resour Announc 2019 Nov 7;8(45). Epub 2019 Nov 7.

Genetics, Faculty of Biology, Ludwig-Maximilians-University Munich, Munich, Germany

We report here the draft genome sequence of mTS5, isolated from a root nodule. The genome consists of 5,454,168 bp, with a GC content of 57%, and contains 5,676 protein-coding sequences.
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http://dx.doi.org/10.1128/MRA.00968-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838617PMC
November 2019

Phenotypic Heterogeneity of the Insect Pathogen Photorhabdus luminescens: Insights into the Fate of Secondary Cells.

Appl Environ Microbiol 2019 11 30;85(22). Epub 2019 Oct 30.

Institut für Molekulare Physiologie, Mikrobiologie und Weinforschung, Johannes-Gutenberg-Universität Mainz, Mainz, Germany

is a Gram-negative bacterium that lives in symbiosis with soil nematodes and is simultaneously highly pathogenic toward insects. The bacteria exist in two phenotypically different forms, designated primary (1°) and secondary (2°) cells. Yet unknown environmental stimuli as well as global stress conditions induce phenotypic switching of up to 50% of 1° cells to 2° cells. An important difference between the two phenotypic forms is that 2° cells are unable to live in symbiosis with nematodes and are therefore believed to remain in the soil after a successful infection cycle. In this work, we performed a transcriptomic analysis to highlight and better understand the role of 2° cells and their putative ability to adapt to living in soil. We could confirm that the major phenotypic differences between the two cell forms are mediated at the transcriptional level as the corresponding genes were downregulated in 2° cells. Furthermore, 2° cells seem to be adapted to another environment as we found several differentially expressed genes involved in the cells' metabolism, motility, and chemotaxis as well as stress resistance, which are either up- or downregulated in 2° cells. As 2° cells, in contrast to 1° cells, chemotactically responded to different attractants, including plant root exudates, there is evidence for the rhizosphere being an alternative environment for the 2° cells. Since is biotechnologically used as a bio-insecticide, investigation of a putative interaction of 2° cells with plants is also of great interest for agriculture. The biological function and the fate of 2° cells were unclear. Here, we performed comparative transcriptomics of 1° and 2° cultures and found several genes, not only those coding for known phenotypic differences of the two cell forms, that are up- or downregulated in 2° cells compared to levels in 1° cells. Our results suggest that when 1° cells convert to 2° cells, they drastically change their way of life. Thus, 2° cells could easily adapt to an alternative environment such as the rhizosphere and live freely, independent of a host, putatively utilizing plant-derived compounds as nutrient sources. Since 2° cells are not able to reassociate with the nematodes, an alternative lifestyle in the rhizosphere would be conceivable.
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http://dx.doi.org/10.1128/AEM.01910-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821960PMC
November 2019

Frankia-Enriched Metagenomes from the Earliest Diverging Symbiotic Frankia Cluster: They Come in Teams.

Genome Biol Evol 2019 08;11(8):2273-2291

Department of Ecology, Environment and Plant Sciences, Stockholm University, Sweden.

Frankia strains induce the formation of nitrogen-fixing nodules on roots of actinorhizal plants. Phylogenetically, Frankia strains can be grouped in four clusters. The earliest divergent cluster, cluster-2, has a particularly wide host range. The analysis of cluster-2 strains has been hampered by the fact that with two exceptions, they could never be cultured. In this study, 12 Frankia-enriched metagenomes of Frankia cluster-2 strains or strain assemblages were sequenced based on seven inoculum sources. Sequences obtained via DNA isolated from whole nodules were compared with those of DNA isolated from fractionated preparations enhanced in the Frankia symbiotic structures. The results show that cluster-2 inocula represent groups of strains, and that strains not represented in symbiotic structures, that is, unable to perform symbiotic nitrogen fixation, may still be able to colonize nodules. Transposase gene abundance was compared in the different Frankia-enriched metagenomes with the result that North American strains contain more transposase genes than Eurasian strains. An analysis of the evolution and distribution of the host plants indicated that bursts of transposition may have coincided with niche competition with other cluster-2 Frankia strains. The first genome of an inoculum from the Southern Hemisphere, obtained from nodules of Coriaria papuana in Papua New Guinea, represents a novel species, postulated as Candidatus Frankia meridionalis. All Frankia-enriched metagenomes obtained in this study contained homologs of the canonical nod genes nodABC; the North American genomes also contained the sulfotransferase gene nodH, while the genome from the Southern Hemisphere only contained nodC and a truncated copy of nodB.
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http://dx.doi.org/10.1093/gbe/evz153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6735867PMC
August 2019

An RNA Chaperone-Like Protein Plays Critical Roles in Chloroplast mRNA Stability and Translation in Arabidopsis and Maize.

Plant Cell 2019 06 8;31(6):1308-1327. Epub 2019 Apr 8.

Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China

A key characteristic of chloroplast gene expression is the predominance of posttranscriptional control via numerous nucleus-encoded RNA binding factors. Here, we explored the essential roles of the S1-domain-containing protein ()/ Stabilizing Factor (BSF) in the stabilization and translation of chloroplast mRNAs. BSF binds to the intergenic region of -, thereby stabilizing 3' processed transcripts and stimulating translation. BSF also binds to the 5' untranslated region of and activates its translation. BSF displayed nucleic-acid-melting activity in vitro, and its absence induces structural changes to target RNAs in vivo, suggesting that BSF functions as an RNA chaperone to remodel RNA structure. BSF physically interacts with the pentatricopeptide repeat protein Chloroplast RNA Processing 1 (AtCRP1) and the ribosomal release factor-like protein Peptide chain Release Factor 3 (PrfB3), whose established RNA ligands overlap with those of BSF. In addition, PrfB3 stimulated the RNA binding ability of BSF in vitro. We propose that BSF and PrfB3 cooperatively reduce the formation of secondary RNA structures within target mRNAs and facilitate AtCRP1 binding. The translation activation function of BSF for is conserved in Arabidopsis () and maize (), but that for operates specifically in Arabidopsis. Our study sheds light on the mechanisms by which RNA binding proteins cooperatively regulate mRNA stability and translation in chloroplasts.
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http://dx.doi.org/10.1105/tpc.18.00946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588297PMC
June 2019

Smut infection of perennial hosts: the genome and the transcriptome of the Brassicaceae smut fungus Thecaphora thlaspeos reveal functionally conserved and novel effectors.

New Phytol 2019 05 17;222(3):1474-1492. Epub 2019 Feb 17.

Institute for Microbiology, Cluster of Excellence on Plant Sciences, Heinrich-Heine University, Building 26.12.01, Universitätsstr. 1, Düsseldorf, 40225, Germany.

Biotrophic fungal plant pathogens can balance their virulence and form intricate relationships with their hosts. Sometimes, this leads to systemic host colonization over long time scales without macroscopic symptoms. However, how plant-pathogenic endophytes manage to establish their sustained systemic infection remains largely unknown. Here, we present a genomic and transcriptomic analysis of Thecaphora thlaspeos. This relative of the well studied grass smut Ustilago maydis is the only smut fungus adapted to Brassicaceae hosts. Its ability to overwinter with perennial hosts and its systemic plant infection including roots are unique characteristics among smut fungi. The T. thlaspeos genome was assembled to the chromosome level. It is a typical smut genome in terms of size and genome characteristics. In silico prediction of candidate effector genes revealed common smut effector proteins and unique members. For three candidates, we have functionally demonstrated effector activity. One of these, TtTue1, suggests a potential link to cold acclimation. On the plant side, we found evidence for a typical immune response as it is present in other infection systems, despite the absence of any macroscopic symptoms during infection. Our findings suggest that T. thlaspeos distinctly balances its virulence during biotrophic growth ultimately allowing for long-lived infection of its perennial hosts.
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http://dx.doi.org/10.1111/nph.15692DOI Listing
May 2019

Complete genome of Rhizobium Norway, an ineffective micro-symbiont.

Stand Genomic Sci 2018 5;13:36. Epub 2018 Dec 5.

Institute of Genetics, Faculty of Biology, Ludwig-Maximilians-University Munich, Munich, Germany.

Rhizobia bacteria engage in nitrogen-fixing root nodule symbiosis, a mutualistic interaction with legume plants in which a bidirectional nutrient exchange takes place. Occasionally, this interaction is suboptimal resulting in the formation of ineffective nodules in which little or no atmospheric nitrogen fixation occurs. Norway induces ineffective nodules in a wide range of hosts. To investigate the basis of this phenotype, we sequenced the complete genome of Norway and compared it to the genome of the closely related strain bv. 3841. The genome comprises 7,788,085 bp, distributed on a circular chromosome containing 63% of the genomic information and five large circular plasmids. The functionally classified bacterial gene set is distributed evenly among all replicons. All symbiotic genes (, , ) are located on the pRLN3 plasmid. Whole genome comparisons revealed differences in the metabolic repertoire and in protein secretion systems, but not in classical symbiotic genes.
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http://dx.doi.org/10.1186/s40793-018-0336-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6280393PMC
December 2018

Single nucleus sequencing reveals evidence of inter-nucleus recombination in arbuscular mycorrhizal fungi.

Elife 2018 12 5;7. Epub 2018 Dec 5.

Department of Biology, University of Ottawa, Ottawa, Canada.

Eukaryotes thought to have evolved clonally for millions of years are referred to as ancient asexuals. The oldest group among these are the arbuscular mycorrhizal fungi (AMF), which are plant symbionts harboring hundreds of nuclei within one continuous cytoplasm. Some AMF strains (dikaryons) harbor two co-existing nucleotypes but there is no direct evidence that such nuclei recombine in this life-stage, as is expected for sexual fungi. Here, we show that AMF nuclei with distinct genotypes can undergo recombination. Inter-nuclear genetic exchange varies in frequency among strains, and despite recombination all nuclear genomes have an average similarity of at least 99.8%. The present study demonstrates that AMF can generate genetic diversity via meiotic-like processes in the absence of observable mating. The AMF dikaryotic life-stage is a primary source of nuclear variability in these organisms, highlighting its potential for strain enhancement of these symbionts.
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http://dx.doi.org/10.7554/eLife.39813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6281316PMC
December 2018

PUMPKIN, the Sole Plastid UMP Kinase, Associates with Group II Introns and Alters Their Metabolism.

Plant Physiol 2019 01 8;179(1):248-264. Epub 2018 Nov 8.

Plant Sciences, Faculty of Biology, Ludwig-Maximilians-University Munich, Großhaderner Street 2-4, 82152 Planegg-Martinsried, Germany

The chloroplast hosts photosynthesis and a variety of metabolic pathways that are essential for plant viability and acclimation processes. In this study, we show that the sole plastid UMP kinase (PUMPKIN) in Arabidopsis () associates specifically with the introns of the plastid transcripts -UCC, -UAC, and in vivo, as revealed by RNA immunoprecipitation coupled with deep sequencing (RIP-Seq); and that PUMPKIN can bind RNA efficiently in vitro. Analyses of target transcripts showed that PUMPKIN affects their metabolism. Null alleles and knockdowns of were viable but clearly affected in growth, plastid translation, and photosynthetic performance. In mutants, the levels of many plastid transcripts were reduced, while the amounts of others were increased, as revealed by RNA-Seq analysis. PUMPKIN is a homomultimeric, plastid-localized protein that forms in vivo RNA-containing megadalton-sized complexes and catalyzes the ATP-dependent conversion of UMP to UDP in vitro with properties characteristic of known essential eubacterial UMP kinases. A moonlighting function of PUMPKIN combining RNA and pyrimidine metabolism is discussed.
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http://dx.doi.org/10.1104/pp.18.00687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6324238PMC
January 2019

Daily rhythms and enrichment patterns in the transcriptome of the behavior-manipulating parasite Ophiocordyceps kimflemingiae.

PLoS One 2017 3;12(11):e0187170. Epub 2017 Nov 3.

LMU Munich, Institute of Medical Psychology, Faculty of Medicine, Munich, Germany.

Various parasite-host interactions that involve adaptive manipulation of host behavior display time-of-day synchronization of certain events. One example is the manipulated biting behavior observed in Carpenter ants infected with Ophiocordyceps unilateralis sensu lato. We hypothesized that biological clocks play an important role in this and other parasite-host interactions. In order to identify candidate molecular clock components, we used two general strategies: bioinformatics and transcriptional profiling. The bioinformatics approach was used to identify putative homologs of known clock genes. For transcriptional profiling, RNA-Seq was performed on 48 h time courses of Ophiocordyceps kimflemingiae (a recently named species of the O. unilateralis complex), whose genome has recently been sequenced. Fungal blastospores were entrained in liquid media under 24 h light-dark (LD) cycles and were harvested at 4 h intervals either under LD or continuous darkness. Of all O. kimflemingiae genes, 5.3% had rhythmic mRNAs under these conditions (JTK Cycle, ≤ 0.057 statistical cutoff). Our data further indicates that a significant number of transcription factors have a peaked activity during the light phase (day time). The expression levels of a significant number of secreted enzymes, proteases, toxins and small bioactive compounds peaked during the dark phase or subjective night. These findings support a model whereby this fungal parasite uses its biological clock for phase-specific activity. We further suggest that this may be a general mechanism involved in parasite-host interactions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187170PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5669440PMC
November 2017

Ant-infecting Ophiocordyceps genomes reveal a high diversity of potential behavioral manipulation genes and a possible major role for enterotoxins.

Sci Rep 2017 10 2;7(1):12508. Epub 2017 Oct 2.

Pennsylvania State University, Entomology and Biology Departments, University, Park, PA, 16802, USA.

Much can be gained from revealing the mechanisms fungal entomopathogens employ. Especially intriguing are fungal parasites that manipulate insect behavior because, presumably, they secrete a wealth of bioactive compounds. To gain more insight into their strategies, we compared the genomes of five ant-infecting Ophiocordyceps species from three species complexes. These species were collected across three continents, from five different ant species in which they induce different levels of manipulation. A considerable number of (small) secreted and pathogenicity-related proteins were only found in these ant-manipulating Ophiocordyceps species, and not in other ascomycetes. However, few of those proteins were conserved among them, suggesting that several different methods of behavior modification have evolved. This is further supported by a relatively fast evolution of previously reported candidate manipulation genes associated with biting behavior. Moreover, secondary metabolite clusters, activated during biting behavior, appeared conserved within a species complex, but not beyond. The independent co-evolution between these manipulating parasites and their respective hosts might thus have led to rather diverse strategies to alter behavior. Our data indicate that specialized, secreted enterotoxins may play a major role in one of these strategies.
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http://dx.doi.org/10.1038/s41598-017-12863-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5624889PMC
October 2017

A quantitative hypermorphic allele confers ectopic calcium flux and impairs cellular development.

Elife 2017 09 21;6. Epub 2017 Sep 21.

Faculty of Biology, Institute of Genetics, Ludwig Maximilian University of Munich, Munich, Germany.

The coordinated control of Ca signaling is essential for development in eukaryotes. Cyclic nucleotide-gated channel (CNGC) family members mediate Ca influx from cellular stores in plants (Charpentier et al., 2016; Gao et al., 2016; Frietsch et al., 2007; Urquhart et al., 2007). Here, we report the unusual genetic behavior of a quantitative gain-of-function mutation () in resulting in a leaky tetrameric channel. resides in a cluster of redundant encoding subunits which resemble metazoan voltage-gated potassium (Kv1-Kv4) channels in assembly and gating properties. The recessive mongenic mutation impaired root development and infection by nitrogen-fixing rhizobia. The allele exhibited quantitative behavior since overexpression of the cluster subunits was required to suppress the phenotype. The results reveal a mechanism by which quantitative competition between channel subunits for tetramer assembly can impact the phenotype of the mutation carrier.
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http://dx.doi.org/10.7554/eLife.25012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716663PMC
September 2017

PALE CRESS binds to plastid RNAs and facilitates the biogenesis of the 50S ribosomal subunit.

Plant J 2017 Nov 21;92(3):400-413. Epub 2017 Sep 21.

Plant Sciences, Faculty of Biology, Ludwig-Maximilians-University Munich, Großhaderner Straße 2-4, 82152, Planegg-Martinsried, Germany.

The plant-specific PALE CRESS (PAC) protein has previously been shown to be essential for photoautotrophic growth. Here we further investigated the molecular function of the PAC protein. PAC localizes to plastid nucleoids and forms large proteinaceous and RNA-containing megadalton complexes. It co-immunoprecipitates with a specific subset of chloroplast RNAs including psbK-psbI, ndhF, ndhD, and 23S ribosomal RNA (rRNA), as demonstrated by RNA immunoprecipitation in combination with high throughput RNA sequencing (RIP-seq) analyses. Furthermore, it co-migrates with premature 50S ribosomal particles and specifically binds to 23S rRNA in vitro. This coincides with severely reduced levels of 23S rRNA in pac leading to translational deficiencies and related alterations of plastid transcript patterns and abundance similar to plants treated with the translation inhibitor lincomycin. Thus, we conclude that deficiency in plastid ribosomes accounts for the pac phenotype. Moreover, the absence or reduction of PAC levels in the corresponding mutants induces structural changes of the 23S rRNA, as demonstrated by in vivo RNA structure probing. Our results indicate that PAC binds to the 23S rRNA to promote the biogenesis of the 50S subunit.
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http://dx.doi.org/10.1111/tpj.13662DOI Listing
November 2017

Whole-Genome Sequences of 14 Strains of and 1 Strain of Isolated from spp. in Algeria.

Genome Announc 2017 Jul 20;5(29). Epub 2017 Jul 20.

Plants and Pathogens Group, Research Institute Land Nature and Environment, hepia, HES-SO University of Applied Sciences and Arts Western Switzerland, Jussy, Geneva, Switzerland

We report here the whole-genome sequences of 14 strains of , isolated from root nodules of and , and 1 strain of isolated from root nodules from in Algeria. These sequences add to the known diversity of this agronomically important genus.
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http://dx.doi.org/10.1128/genomeA.00676-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5522942PMC
July 2017

The Plant-Dependent Life Cycle of Thecaphora thlaspeos: A Smut Fungus Adapted to Brassicaceae.

Mol Plant Microbe Interact 2017 04 19;30(4):271-282. Epub 2017 Apr 19.

1 Institute for Microbiology, Cluster of Excellence in Plant Sciences, Heinrich-Heine University, Building 26.12.01, Universitätsstr.1, 40205 Düsseldorf, Germany.

Smut fungi are globally distributed plant pathogens that infect agriculturally important crop plants such as maize or potato. To date, molecular studies on plant responses to smut fungi are challenging due to the genetic complexity of their host plants. Therefore, we set out to investigate the known smut fungus of Brassicaceae hosts, Thecaphora thlaspeos. T. thlaspeos infects different Brassicaceae plant species throughout Europe, including the perennial model plant Arabis alpina. In contrast to characterized smut fungi, mature and dry T. thlaspeos teliospores germinated only in the presence of a plant signal. An infectious filament emerges from the teliospore, which can proliferate as haploid filamentous cultures. Haploid filaments from opposite mating types mate, similar to sporidia of the model smut fungus Ustilago maydis. Consistently, the a and b mating locus genes are conserved. Infectious filaments can penetrate roots and aerial tissues of host plants, causing systemic colonization along the vasculature. Notably, we could show that T. thlaspeos also infects Arabidopsis thaliana. Exploiting the genetic resources of A. thaliana and Arabis alpina will allow us to characterize plant responses to smut infection in a comparative manner and, thereby, characterize factors for endophytic growth as well as smut fungi virulence in dicot plants.
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http://dx.doi.org/10.1094/MPMI-08-16-0164-RDOI Listing
April 2017

Revisiting regulation of potassium homeostasis in Escherichia coli: the connection to phosphate limitation.

Microbiologyopen 2017 06 17;6(3). Epub 2017 Jan 17.

Fachbereich Biologie/Chemie, Universität Osnabrück, Osnabrück, Germany.

Two-component signal transduction constitutes the predominant strategy used by bacteria to adapt to fluctuating environments. The KdpD/KdpE system is one of the most widespread, and is crucial for K homeostasis. In Escherichia coli, the histidine kinase KdpD senses K availability, whereas the response regulator KdpE activates synthesis of the high-affinity K uptake system KdpFABC. Here we show that, in the absence of KdpD, kdpFABC expression can be activated via phosphorylation of KdpE by the histidine kinase PhoR. PhoR and its cognate response regulator PhoB comprise a phosphate-responsive two-component system, which senses phosphate limitation indirectly through the phosphate transporter PstCAB and its accessory protein PhoU. In vivo two-hybrid interaction studies based on the bacterial adenylate cyclase reveal pairwise interactions between KdpD, PhoR, and PhoU. Finally, we demonstrate that cross-regulation between the kdpFABC and pstSCAB operons occurs in both directions under simultaneous K and phosphate limitation, both in vitro and in vivo. This study for the first time demonstrates direct coupling between intracellular K and phosphate homeostasis and provides a mechanism for fine-tuning of the balance between positively and negatively charged ions in the bacterial cell.
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http://dx.doi.org/10.1002/mbo3.438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458449PMC
June 2017

Fungal Mating in the Most Widespread Plant Symbionts?

Trends Plant Sci 2017 02 19;22(2):175-183. Epub 2016 Nov 19.

LMU Munich, Faculty of Biology, Genetics, D-82152 Planegg-Martinsried, Germany.

Arbuscular mycorrhizal fungi (AMF) are relevant plant symbionts whose hyphae and spores carry hundreds of coexisting nuclei with supposedly divergent genomes but no sign of sexual reproduction. This unusual biology suggested that conventional fungal mating is not amendable to optimize strains for plant growth, but recent evidence of sexual-related nuclear inheritance in these organisms is now challenging this widespread notion. Here, we outline our knowledge of AMF genetics within a historical context, and discuss how past and new information in this area changed our understanding of AMF biology. We also highlight the mating-related processes in AMF, and propose new research avenues and approaches that could lead to a better application of these organisms for agricultural and environmental practices.
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http://dx.doi.org/10.1016/j.tplants.2016.10.010DOI Listing
February 2017

Evidence for the sexual origin of heterokaryosis in arbuscular mycorrhizal fungi.

Nat Microbiol 2016 03 21;1(6):16033. Epub 2016 Mar 21.

Canadian Institute for Advanced Research, Department of Biology, University of Ottawa, Ottawa, Ontario, Canada.

Sexual reproduction is ubiquitous among eukaryotes, and fully asexual lineages are extremely rare. Prominent among ancient asexual lineages are the arbuscular mycorrhizal fungi (AMF), a group of plant symbionts with a multinucleate cytoplasm. Genomic divergence among co-existing nuclei was proposed to drive the evolutionary success of AMF in the absence of sex(1), but this hypothesis has been contradicted by recent genome analyses that failed to find significant genetic diversity within an AMF isolate(2,3). Here, we set out to resolve issues surrounding the genome organization and sexual potential of AMF by exploring the genomes of five isolates of Rhizophagus irregularis, a model AMF. We find that genetic diversity in this species varies among isolates and is structured in a homo-dikaryon-like manner usually linked with the existence of a sexual life cycle. We also identify a putative AMF mating-type locus, containing two genes with structural and evolutionary similarities with the mating-type locus of some Dikarya. Our analyses suggest that this locus may be multi-allelic and that AMF could be heterothallic and bipolar. These findings reconcile opposing views on the genome organization of these ubiquitous plant symbionts and open avenues for strain improvement and environmental application of these organisms.
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http://dx.doi.org/10.1038/nmicrobiol.2016.33DOI Listing
March 2016

GintAMT3 - a Low-Affinity Ammonium Transporter of the Arbuscular Mycorrhizal Rhizophagus irregularis.

Front Plant Sci 2016 25;7:679. Epub 2016 May 25.

Department of Environmental Sciences, Botany, Zurich-Basel Plant Science Center, University of Basel Basel, Switzerland.

Nutrient acquisition and transfer are essential steps in the arbuscular mycorrhizal (AM) symbiosis, which is formed by the majority of land plants. Mineral nutrients are taken up by AM fungi from the soil and transferred to the plant partner. Within the cortical plant root cells the fungal hyphae form tree-like structures (arbuscules) where the nutrients are released to the plant-fungal interface, i.e., to the periarbuscular space, before being taken up by the plant. In exchange, the AM fungi receive carbohydrates from the plant host. Besides the well-studied uptake of phosphorus (P), the uptake and transfer of nitrogen (N) plays a crucial role in this mutualistic interaction. In the AM fungus Rhizophagus irregularis (formerly called Glomus intraradices), two ammonium transporters (AMT) were previously described, namely GintAMT1 and GintAMT2. Here, we report the identification and characterization of a newly identified R. irregularis AMT, GintAMT3. Phylogenetic analyses revealed high sequence similarity to previously identified AM fungal AMTs and a clear separation from other fungal AMTs. Topological analysis indicated GintAMT3 to be a membrane bound pore forming protein, and GFP tagging showed it to be highly expressed in the intraradical mycelium of a fully established AM symbiosis. Expression of GintAMT3 in yeast successfully complemented the yeast AMT triple deletion mutant (MATa ura3 mep1Δ mep2Δ::LEU2 mep3Δ::KanMX2). GintAMT3 is characterized as a low affinity transport system with an apparent Km of 1.8 mM and a V max of 240 nmol(-1) min(-1) 10(8) cells(-1), which is regulated by substrate concentration and carbon supply.
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http://dx.doi.org/10.3389/fpls.2016.00679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4879785PMC
June 2016

Identification and Initial Characterization of Prophages in Vibrio campbellii.

PLoS One 2016 23;11(5):e0156010. Epub 2016 May 23.

Munich Center for Integrated Protein Science (CiPSM) at the Department of Biology I, Microbiology, Ludwig-Maximilians-Universität München, Martinsried, Germany.

Phages are bacteria targeting viruses and represent the most abundant biological entities on earth. Marine environments are exceptionally rich in bacteriophages, harboring a total of 4x10(30) viruses. Nevertheless, marine phages remain poorly characterized. Here we describe the identification of intact prophage sequences in the genome of the marine γ-proteobacterium Vibrio campbellii ATCC BAA-1116 (formerly known as V. harveyi ATCC BAA-1116), which presumably belong to the family of Myoviridae. One prophage was found on chromosome I and shows significant similarities to the previously identified phage ΦHAP-1. The second prophage region is located on chromosome II and is related to Vibrio phage kappa. Exposure of V. campbellii to mitomycin C induced the lytic cycle of two morphologically distinct phages and, as expected, extracellular DNA from induced cultures was found to be specifically enriched for the sequences previously identified as prophage regions. Heat stress (50°C, 30 min) was also found to induce phage release in V. campbellii. Notably, promoter activity of two representative phage genes indicated heterogeneous phage induction within the population.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0156010PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4877103PMC
July 2017

Diversity and Composition of the Leaf Mycobiome of Beech (Fagus sylvatica) Are Affected by Local Habitat Conditions and Leaf Biochemistry.

PLoS One 2016 14;11(4):e0152878. Epub 2016 Apr 14.

Ruhr-Universität Bochum, AG Geobotanik, Bochum, Germany.

Comparative investigations of plant-associated fungal communities (mycobiomes) in distinct habitats and under distinct climate regimes have been rarely conducted in the past. Nowadays, high-throughput sequencing allows routine examination of mycobiome responses to environmental changes and results at an unprecedented level of detail. In the present study, we analysed Illumina-generated fungal ITS1 sequences from European beech (Fagus sylvatica) originating from natural habitats at two different altitudes in the German Alps and from a managed tree nursery in northern Germany. In general, leaf-inhabiting mycobiome diversity and composition correlated significantly with the origin of the trees. Under natural condition the mycobiome was more diverse at lower than at higher elevation, whereas fungal diversity was lowest in the artificial habitat of the tree nursery. We further identified significant correlation of leaf chlorophylls and flavonoids with both habitat parameters and mycobiome biodiversity. The present results clearly point towards a pronounced importance of local stand conditions for the structure of beech leaf mycobiomes and for a close interrelation of phyllosphere fungi and leaf physiology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152878PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4831807PMC
August 2016

Overproduction of Magnetosomes by Genomic Amplification of Biosynthesis-Related Gene Clusters in a Magnetotactic Bacterium.

Appl Environ Microbiol 2016 05 2;82(10):3032-3041. Epub 2016 May 2.

Ludwig-Maximilians-Universität München, Department of Biology I, Martinsried, Germany

Unlabelled: Magnetotactic bacteria biosynthesize specific organelles, the magnetosomes, which are membrane-enclosed crystals of a magnetic iron mineral that are aligned in a linear chain. The number and size of magnetosome particles have to be critically controlled to build a sensor sufficiently strong to ensure the efficient alignment of cells within Earth's weak magnetic field while at the same time minimizing the metabolic costs imposed by excessive magnetosome biosynthesis. Apart from their biological function, bacterial magnetosomes have gained considerable interest since they provide a highly useful model for prokaryotic organelle formation and represent biogenic magnetic nanoparticles with exceptional properties. However, potential applications have been hampered by the difficult cultivation of these fastidious bacteria and their poor yields of magnetosomes. In this study, we found that the size and number of magnetosomes within the cell are controlled by many different Mam and Mms proteins. We present a strategy for the overexpression of magnetosome biosynthesis genes in the alphaproteobacterium Magnetospirillum gryphiswaldense by chromosomal multiplication of individual and multiple magnetosome gene clusters via transposition. While stepwise amplification of the mms6 operon resulted in the formation of increasingly larger crystals (increase of ∼35%), the duplication of all major magnetosome operons (mamGFDC, mamAB, mms6, and mamXY, comprising 29 genes in total) yielded an overproducing strain in which magnetosome numbers were 2.2-fold increased. We demonstrate that the tuned expression of the mam and mms clusters provides a powerful strategy for the control of magnetosome size and number, thereby setting the stage for high-yield production of tailored magnetic nanoparticles by synthetic biology approaches.

Importance: Before our study, it had remained unknown how the upper sizes and numbers of magnetosomes are genetically regulated, and overproduction of magnetosome biosynthesis had not been achieved, owing to the difficulties of large-scale genome engineering in the recalcitrant magnetotactic bacteria. In this study, we established and systematically explored a strategy for the overexpression of magnetosome biosynthesis genes by genomic amplification of single and multiple magnetosome gene clusters via sequential chromosomal insertion by transposition. Our findings also indicate that the expression levels of magnetosome proteins together limit the upper size and number of magnetosomes within the cell. We demonstrate that tuned overexpression of magnetosome gene clusters provides a powerful strategy for the precise control of magnetosome size and number.
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http://dx.doi.org/10.1128/AEM.03860-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4959066PMC
May 2016