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

ABSTRACT 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 Magnetospirillum gryphiswaldense using a systematic genome-wide transposon mutagenesis approach. By an optimized procedure, a Tn5 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 Tn5 insertion sites mapped within the MAI, mostly leading to a nonmagnetic phenotype. In contrast, in the majority of weakly magnetic Tn5 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 Tn5-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 c maturation, or are altogether of unknown function. We also found that signal transduction and redox functions are enriched in the set of Tn5 hits outside the MAI, suggesting that such processes are particularly important in support of magnetosome biosynthesis. IMPORTANCE Magnetospirillum gryphiswaldense 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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Phenotypic Screen and Transcriptomics Approach Complement Each Other in Functional Genomics of Defensive Stink Gland Physiology

10.21203/rs.3.rs-1117784/v1 ◽

2021 ◽

Author(s):

Sabrina Lehmann ◽

Bibi Atika ◽

Daniela Grossmann ◽

Christian Schmitt-Engel ◽

Nadi Strohlein ◽

...

Keyword(s):

Functional Genomics ◽

Large Scale ◽

Reverse Genetics ◽

Expression Profiles ◽

Forward Genetics ◽

Large Set ◽

Knock Down ◽

Genome Wide ◽

Phenotypic Screen ◽

A Genome

Abstract Background Functional genomics uses unbiased systematic genome-wide gene disruption or analyzes natural variations such as gene expression profiles of different tissues from multicellular organisms to link gene functions to particular phenotypes. Functional genomics approaches are of particular importance to identify large sets of genes that are specifically important for a particular biological process beyond known candidate genes, or when the process has not been studied with genetic methods before. Results Here, we present a large set of genes whose disruption interferes with the function of the odoriferous defensive stink glands of the red flour beetle Tribolium castaneum. This gene set is the result of a large-scale systematic phenotypic screen using a reverse genetics strategy based on RNA interference applied in a genome-wide forward genetics manner. In this first-pass screen, 130 genes were identified, of which 69 genes could be confirmed to cause knock-down gland phenotypes, which vary from necrotic tissue and irregular reservoir size to irregular color or separation of the secreted gland compounds. The knock-down of 13 genes caused specifically a strong reduction of para-benzoquinones, suggesting a specific function in the synthesis of these toxic compounds. Only 14 of the 69 confirmed gland genes are differentially overexpressed in stink gland tissue and thus could have been detected in a transcriptome-based analysis. Moreover, of the 29 previously transcriptomics-identified genes causing a gland phenotype, only one gene was recognized by this phenotypic screen despite the fact that 13 of them were covered by the screen. Conclusion Our results indicate the importance of combining diverse and independent methodologies to identify genes necessary for the function of a certain biological tissue, as the different approaches do not deliver redundant results but rather complement each other. The presented phenotypic screen together with a transcriptomics approach are now providing a set of close to hundred genes important for odoriferous defensive stink gland physiology in beetles.

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Bacterioferritin ofMagnetospirillum gryphiswaldenseIs a Heterotetraeicosameric Complex Composed of Functionally Distinct Subunits but Is Not Involved in Magnetite Biomineralization

mBio ◽

10.1128/mbio.02795-18 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 4

Author(s):

René Uebe ◽

Frederik Ahrens ◽

Jörg Stang ◽

Katharina Jäger ◽

Lars H. Böttger ◽

...

Keyword(s):

Oxidative Stress ◽

Magnetotactic Bacteria ◽

Functional Differentiation ◽

Spectroscopic Techniques ◽

Content Type ◽

Magnetospirillum Gryphiswaldense ◽

Ferroxidase Activity ◽

Magnetite Biomineralization ◽

Field Lines

ABSTRACTThe biomineralization pathway of magnetite in magnetotactic bacteria is still poorly understood and a matter of intense debates. In particular, the existence, nature, and location of possible mineral precursors of magnetite are not clear. One possible precursor has been suggested to be ferritin-bound ferrihydrite. To clarify its role for magnetite biomineralization, we analyzed and characterized ferritin-like proteins from the magnetotactic alphaproteobacteriumMagnetospirillum gryphiswaldenseMSR-1, employing genetic, biochemical, and spectroscopic techniques. Transmission Mössbauer spectroscopy of the wild type (WT) and a bacterioferritin (bfr) deletion strain uncovered that the presence of ferrihydrite in cells is coupled to the presence of Bfr. However,bfranddpsdeletion mutants, encoding another ferritin-like protein, or even mutants with their codeletion had no impact on magnetite formation in MSR-1. Thus, ferritin-like proteins are not involved in magnetite biomineralization and Bfr-bound ferrihydrite is not a precursor of magnetite biosynthesis. Using transmission electron microscopy and bacterial two-hybrid and electrophoretic methods, we also show that MSR-1 Bfr is an atypical representative of the Bfr subfamily, as it forms tetraeicosameric complexes from two distinct subunits. Furthermore, our analyses revealed that these subunits are functionally divergent, with Bfr1 harboring a ferroxidase activity while only Bfr2 contributes to heme binding. Because of this functional differentiation and the poor formation of homooligomeric Bfr1 complexes, only heterooligomeric Bfr protects cells from oxidative stressin vivo.In summary, our results not only provide novel insights into the biomineralization of magnetite but also reveal the unique properties of so-far-uncharacterized heterooligomeric bacterioferritins.IMPORTANCEMagnetotactic bacteria likeMagnetospirillum gryphiswaldenseare able to orient along magnetic field lines due to the intracellular formation of magnetite nanoparticles. Biomineralization of magnetite has been suggested to require a yet-unknown ferritin-like ferrihydrite component. Here, we report the identification of a bacterioferritin as the source of ferrihydrite inM. gryphiswaldenseand show that, contrary to previous reports, bacterioferritin is not involved in magnetite biomineralization but required for oxidative stress resistance. Additionally, we show that bacterioferritin ofM. gryphiswaldenseis an unusual member of the bacterioferritin subfamily as it is composed of two functionally distinct subunits. Thus, our findings extend our understanding of the bacterioferritin subfamily and also solve a longstanding question about the magnetite biomineralization pathway.

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Rapid Quantification of Mutant Fitness in Diverse Bacteria by Sequencing Randomly Bar-Coded Transposons

mBio ◽

10.1128/mbio.00306-15 ◽

2015 ◽

Vol 6 (3) ◽

Cited By ~ 167

Author(s):

Kelly M. Wetmore ◽

Morgan N. Price ◽

Robert J. Waters ◽

Jacob S. Lamson ◽

Jennifer He ◽

...

Keyword(s):

Next Generation Sequencing ◽

Biological Significance ◽

Transposon Mutagenesis ◽

Next Generation ◽

Experimental Conditions ◽

Content Type ◽

Bar Code ◽

Genome Wide ◽

Powerful Approach ◽

Generation Sequencing

ABSTRACT Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with any transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative d-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. IMPORTANCE A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.

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FORWARD AND UNBIASED RNA-INTERFERENCE SCREEN

10.31219/osf.io/8y7s4 ◽

2021 ◽

Author(s):

Wadim J Kapulkin

Keyword(s):

Rna Interference ◽

Caenorhabditis Elegans ◽

Reverse Genetics ◽

Screening Strategy ◽

Rnai Screen ◽

Forward Genetics ◽

Genome Wide ◽

Complementary Resource ◽

Genetics Screening ◽

Caenorhabditis Elegans Genome

RNA-interference (Fire et al. 1998) is a popular ‘reverse-genetics’ screening strategy applied in Caenorhabditis elegans. Genome-wide RNAi screens are presently carried using RNAi feeding libraries. Here, we report on a complementary resource facilitating an approach to RNAi screen relying on an unbiased ‘forward-genetics’ strategy. We conclude the forward RNA interference screening is useful and feasible, with the strong expectation the presented screening mode will complement and extend on the existing, currently available, genome-wide RNAi resources.

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Genome-Wide Identification of Fitness Factors in Seawater for Edwardsiella piscicida

Applied and Environmental Microbiology ◽

10.1128/aem.00233-19 ◽

2019 ◽

Vol 85 (10) ◽

Cited By ~ 3

Author(s):

Lifan Wei ◽

Yanyan Wu ◽

Guanhua Yang ◽

Rongjing Xu ◽

Xiaohong Liu ◽

...

Keyword(s):

Type Iii Secretion ◽

Sequencing Analysis ◽

Genetic Determinants ◽

Farmed Fish ◽

Content Type ◽

Transposon Insertion ◽

Genome Wide ◽

Edwardsiella Piscicida ◽

Higher Temperature ◽

Transposon Insertion Sequencing

ABSTRACT Marine pathogens are transmitted from one host to another through seawater. Therefore, it is important for marine pathogens to maintain survival or growth in seawater. However, little is known about how marine pathogens adapt to living in seawater environments. Here, transposon insertion sequencing was performed to explore the genetic determinants of Edwardsiella piscicida survival in seawater at 16 and 28°C. Seventy-one mutants with mutations mainly in metabolism-, transportation-, and type III secretion system (T3SS)-related genes showed significantly increased or impaired fitness in 16°C water. In 28°C seawater, 63 genes associated with transcription and translation, as well as energy production and conversion, were essential for optimal survival of the bacterium. In particular, 11 T3SS-linked mutants displayed enhanced fitness in 16°C seawater but not in 28°C seawater. In addition, 13 genes associated with oxidative phosphorylation and 4 genes related to ubiquinone synthesis were identified for survival in 28°C seawater but not in 16°C seawater, which suggests that electron transmission and energy-producing aerobic respiration chain factors are indispensable for E. piscicida to maintain survival in higher-temperature seawater. In conclusion, we defined genes and processes related to metabolism and virulence that operate in E. piscicida to facilitate survival in low- and high-temperature seawater, which may underlie the infection outbreak mechanisms of E. piscicida and facilitate the development of improved vaccines against marine pathogens. IMPORTANCE Edwardsiella piscicida is one of the most important marine pathogens and causes serious edwardsiellosis in farmed fish during the summer-autumn seasonal changes, resulting in enormous losses to aquaculture industries worldwide. Survival and transmission of the pathogen in seawater are critical steps that increase the risk of outbreaks. To investigate the mechanism of survival in seawater for this marine pathogen, we used transposon insertion sequencing analysis to explore the fitness determinants in summer and autumn seawater. Approximately 127 genes linked to metabolism and virulence, as well as other processes, were revealed in E. piscicida to contribute to better adaptations to the seasonal alternations of seawater environments; these genes provide important insights into the infection outbreak mechanisms of E. piscicida and potential improved treatments or vaccines against marine pathogens.

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The Polar Organizing Protein PopZ Is Fundamental for Proper Cell Division and Segregation of Cellular Content in Magnetospirillum gryphiswaldense

mBio ◽

10.1128/mbio.02716-18 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 7

Author(s):

Daniel Pfeiffer ◽

Mauricio Toro-Nahuelpan ◽

Marc Bramkamp ◽

Jürgen M. Plitzko ◽

Dirk Schüler

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Caulobacter Crescentus ◽

Magnetotactic Bacteria ◽

Model Organisms ◽

Chromosomal Dna ◽

Content Type ◽

Magnetospirillum Gryphiswaldense ◽

Cellular Content ◽

Subcellular Organization

ABSTRACT Magnetotactic bacteria (MTB) are of special scientific interest due to the formation of magnetosomes, intracellular membrane-enveloped magnetite crystals arranged into a linear chain by a dedicated cytoskeleton. Magnetotaxis relies on the formation and proper inheritance of these unique magnetic organelles, both of which need to be coordinated with the segregation of other cellular content such as chromosomes or motility and chemotaxis related structures. Thus, elaborated mechanisms are required in MTB to coordinate and maintain a high level of spatial and temporal subcellular organization during cytokinesis. However, thus far, underlying mechanisms and polarity determinants such as landmark proteins remained obscure in MTB. Here, we analyzed an ortholog of the polar organizing protein Z in the alphaproteobacterium Magnetospirillum gryphiswaldense termed PopZMgr. We show that deletion of the popZMgr gene causes abnormal cell elongation, minicell formation, DNA missegregation, and impairs motility. Overproduction of PopZMgr results in PopZ-rich regions near the poles, which are devoid of larger macromolecules, such as ribosomes, chromosomal DNA, and polyhydroxybutyrate (PHB) granules. Using superresolution microscopy, we show that PopZMgr exhibits a bipolar localization pattern throughout the cell cycle, indicating that the definition of new poles in M. gryphiswaldense occurs immediately upon completion of cytokinesis. Moreover, substitution of PopZ orthologs between M. gryphiswaldense and the related alphaproteobacterium Caulobacter crescentus indicated that PopZ localization depends on host-specific cues and that both orthologs have diverged to an extent that allows only partial reciprocal functional complementation. Altogether, our results indicate that in M. gryphiswaldense, PopZ plays a critical role during cell division and segregation of cellular content. IMPORTANCE Magnetotactic bacteria (MTB) share the unique capability of magnetic navigation, one of the most complex behavioral responses found in prokaryotes, by means of magnetosomes, which act as an internal compass. Due to formation of these unique nanoparticles, MTB have emerged as a model to study prokaryotic organelle formation and cytoskeletal organization in conjunction with complex motility systems. Despite the high degree of subcellular organization required in MTB, less is known about cell-cycle-related factors or proteins responsible for spatiotemporal polarity control. Here, we investigate the function of the polar organizer PopZ in the magnetotactic alphaproteobacterium Magnetospirillum gryphiswaldense. Although PopZ is widely distributed among the alphaproteobacteria, its function in MTB belonging to this class has remained unexplored. Our results suggest that in M. gryphiswaldense, PopZ has a key role during cell division and subcellular organization. Furthermore, we show that PopZ localization and function differ from other nonmagnetotactic alphaproteobacterial model organisms.

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Expression regulation of myo-inositol 3-phosphate synthase 1 (INO1) in determination of phytic acid accumulation in rice grain

Scientific Reports ◽

10.1038/s41598-019-51485-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Ishara Perera ◽

Ayaka Fukushima ◽

Tatsuki Akabane ◽

Genki Horiguchi ◽

Saman Seneweera ◽

...

Keyword(s):

Gene Expression ◽

Phytic Acid ◽

Reverse Genetics ◽

Developmental Stages ◽

Rice Grain ◽

Genetic Determinants ◽

Genome Wide ◽

Significant Single Nucleotide Polymorphism ◽

Early Developmental ◽

Phosphate Synthase

Abstract Phytic acid (PA) is the primary phosphorus (P) storage compound in the seeds of cereals and legumes. Low PA crops, which are considered an effective way to improve grain nutrient availability and combat environmental issues relating to seed P have been developed using mutational and reverse genetics approaches. Here, we identify molecular mechanism regulating PA content among natural rice variants. First, we performed genome-wide association (GWA) mapping of world rice core collection (WRC) accessions to understand the genetic determinants underlying PA trait in rice. Further, a comparative study was undertaken to identify the differences in PA accumulation, protein profiles, and gene expression in low (WRC 5) and high PA (WRC 6) accessions. GWA results identified myo-inositol 3-phosphate synthase 1 (INO1) as being closely localized to a significant single nucleotide polymorphism. We found high rates of PA accumulation 10 days after flowering, and our results indicate that INO1 expression was significantly higher in WRC 6 than in WRC 5. Seed proteome assays found that the expression of INO1 was significantly higher in WRC 6. These results suggest that not only the gene itself but regulation of INO1 gene expression at early developmental stages is important in determining PA content in rice.

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The Periplasmic Nitrate Reductase Nap Is Required for Anaerobic Growth and Involved in Redox Control of Magnetite Biomineralization in Magnetospirillum gryphiswaldense

Journal of Bacteriology ◽

10.1128/jb.00903-12 ◽

2012 ◽

Vol 194 (18) ◽

pp. 4847-4856 ◽

Cited By ~ 55

Author(s):

Yingjie Li ◽

Emanuel Katzmann ◽

Sarah Borg ◽

Dirk Schüler

Keyword(s):

Nitrate Reductase ◽

Mixed Valence ◽

Anaerobic Respiration ◽

Redox Balance ◽

Anaerobic Growth ◽

Content Type ◽

Magnetospirillum Gryphiswaldense ◽

Periplasmic Nitrate Reductase ◽

Magnetite Biomineralization ◽

Denitrification Genes

ABSTRACTThe magnetosomes of many magnetotactic bacteria consist of membrane-enveloped magnetite crystals, whose synthesis is favored by a low redox potential. However, the cellular redox processes governing the biomineralization of the mixed-valence iron oxide have remained unknown. Here, we show that in the alphaproteobacteriumMagnetospirillum gryphiswaldense, magnetite biomineralization is linked to dissimilatory nitrate reduction. A complete denitrification pathway, including gene functions for nitrate (nap), nitrite (nir), nitric oxide (nor), and nitrous oxide reduction (nos), was identified. TranscriptionalgusAfusions as reporters revealed that except fornap, the highest expression of the denitrification genes coincided with conditions permitting maximum magnetite synthesis. Whereas microaerobic denitrification overlapped with oxygen respiration, nitrate was the only electron acceptor supporting growth in the entire absence of oxygen, and only the deletion ofnapgenes, encoding a periplasmic nitrate reductase, and not deletion ofnorornosgenes, abolished anaerobic growth and also delayed aerobic growth in both nitrate and ammonium media. While loss ofnosZornorCBhad no or relatively weak effects on magnetosome synthesis, deletion ofnapseverely impaired magnetite biomineralization and resulted in fewer, smaller, and irregular crystals during denitrification and also microaerobic respiration, probably by disturbing the proper redox balance required for magnetite synthesis. In contrast to the case for the wild type, biomineralization in Δnapcells was independent of the oxidation state of carbon substrates. Altogether, our data demonstrate that in addition to its essential role in anaerobic respiration, the periplasmic nitrate reductase Nap has a further key function by participating in redox reactions required for magnetite biomineralization.

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