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Berlin Brandenburg

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  • 1
    Language: English
    In: Current Opinion in Microbiology, 2011, Vol.14(5), pp.579-586
    Description: ► Technological advances are driving progress in prokaryotic transcriptomics. ► Two concepts, complex, population based and single cell analyses are presented. ► Single cell analyses will alter views concerning transcriptome content and dynamics. ► Global transcriptomic studies involving microbe and plant interactions are lacking. Genome-wide expression studies transformed the field of transcriptomics and made it feasible to study global gene expression in extraordinary detail. These new methods have revealed an enhanced view of the transcriptional landscape and have yielded many biological insights. It is increasingly clear that the prokaryotic transcriptome is much more complex than once thought. Recent advances in microbial transcriptome analyses are highlighted in this review. Areas of progress include the development of optimized techniques that minimize the abundance of ribosomal RNAs in RNA samples as well as the development of novel methods to create transcriptome libraries. Advances such as these have led to a new emphasis in areas such as metatranscriptomics and single cell gene expression studies.
    Keywords: Biology
    ISSN: 1369-5274
    E-ISSN: 1879-0364
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  • 2
    Language: English
    In: Journal of Bacteriology, Sept, 2011, Vol.193(17-18), p.4598(14)
    Description: The plant pathogen Pseudomonas syringae pv. tomato DC3000 (DC3000) is found in a wide variety of environments and must monitor and respond to various environmental signals such as the availability of iron, an essential element for bacterial growth. An important regulator of iron homeostasis is Fur (ferric uptake regulator), and here we present the first study of the Fur regulon in DC3000. Using chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq), 312 chromosomal regions were highly enriched by coimmunoprecipitation with a C-terminally tagged Fur protein. Integration of these data with previous microarray and global transcriptome analyses allowed us to expand the putative DC3000 Fur regulon to include genes both repressed and activated in the presence of bioavailable iron. Using nonradioactive DNase I footprinting, we confirmed Fur binding in 41 regions, including upstream of 11 iron-repressed genes and the iron-activated genes encoding two bacterioferritins (PSPTO_0653 and PSPTO_4160), a ParA protein (PSPTO_0855), and a two-component system (TCS) (PSPTO_3382 to PSPTO_3380). doi: 10.1128/JB.00340-11
    Keywords: Pseudomonas Syringae -- Genetic Aspects ; Pseudomonas Syringae -- Physiological Aspects ; Pseudomonas Syringae -- Research ; Chromatin -- Physiological Aspects ; Chromatin -- Research ; Tomato Breeding -- Research
    ISSN: 0021-9193
    Source: Cengage Learning, Inc.
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  • 3
    Language: English
    In: Journal of bacteriology, 01 March 2018, Vol.200(5)
    Description: Two-component systems (TCSs) of bacteria regulate many different aspects of the bacterial life cycle, including pathogenesis. Most TCSs remain uncharacterized, with no information about the signal(s) or regulatory targets and/or role in bacterial pathogenesis. Here, we characterized a TCS in the plant-pathogenic bacterium pv. tomato DC3000 composed of the histidine kinase CvsS and the response regulator CvsR. CvsSR is necessary for virulence of pv. tomato DC3000, since Δ and Δ strains produced fewer symptoms than the wild type (WT) and demonstrated reduced growth on multiple hosts. We discovered that expression of is induced by Ca concentrations found in leaf apoplastic fluid. Thus, Ca can be added to the list of signals that promote pathogenesis of pv. tomato DC3000 during host colonization. Through chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) and global transcriptome analysis (RNA-seq), we discerned the CvsR regulon. CvsR directly activated expression of the type III secretion system regulators, and , that regulate pv. tomato DC3000 virulence in a type III secretion system-dependent manner. CvsR also indirectly repressed transcription of the extracytoplasmic sigma factor and production of alginate. Phenotypic analysis determined that CvsSR inversely regulated biofilm formation, swarming motility, and cellulose production in a Ca-dependent manner. Overall, our results show that CvsSR is a key regulatory hub critical for interaction with host plants. Pathogenic bacteria must be able to react and respond to the surrounding environment, make use of available resources, and avert or counter host immune responses. Often, these abilities rely on two-component systems (TCSs) composed of interacting proteins that modulate gene expression. We identified a TCS in the plant-pathogenic bacterium that responds to the presence of calcium, which is an important signal during the plant defense response. We showed that when is grown in the presence of calcium, this TCS regulates expression of factors contributing to disease. Overall, our results provide a better understanding of how bacterial pathogens respond to plant signals and control systems necessary for eliciting disease.
    Keywords: Pseudomonas Syringae ; Alginate ; Biofilms ; Calcium Signaling ; Two-Component Regulatory Systems
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 4
    Language: English
    In: PLoS ONE, 2011, Vol.6(12), p.e29335
    Description: RNA-Seq has provided valuable insights into global gene expression in a wide variety of organisms. Using a modified RNA-Seq approach and Illumina's high-throughput sequencing technology, we globally identified 5′-ends of transcripts for the plant pathogen Pseudomonas syringae pv. tomato str. DC3000. A substantial fraction of 5′-ends obtained by this method were consistent with results obtained using global RNA-Seq and 5′RACE. As expected, many 5′-ends were positioned a short distance upstream of annotated genes. We also captured 5′-ends within intergenic regions, providing evidence for the expression of un-annotated genes and non-coding RNAs, and detected numerous examples of antisense transcription, suggesting additional levels of complexity in gene regulation in DC3000. Importantly, targeted searches for sequence patterns in the vicinity of 5′-ends revealed over 1200 putative promoters and other regulatory motifs, establishing a broad foundation for future investigations of regulation at the genomic and single gene levels.
    Keywords: Research Article ; Biology ; Genetics And Genomics ; Microbiology ; Computational Biology
    E-ISSN: 1932-6203
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  • 5
    Language: English
    In: PLoS ONE, 01 January 2013, Vol.8(2), p.e55594
    Description: Pseudomonas aeruginosa (Pae) is a clinically important opportunistic pathogen. Herein, we demonstrate that the PA1006 protein is critical for all nitrate reductase activities, growth as a biofilm in a continuous flow system, as well as virulence in mouse burn and rat lung model systems. Microarray analysis revealed that ΔPA1006 cells displayed extensive alterations in gene expression including nitrate-responsive, quorum sensing (including PQS production), and iron-regulated genes, as well as molybdenum cofactor and Fe-S cluster biosynthesis factors, members of the TCA cycle, and Type VI Secretion System components. Phenotype Microarray™ profiles of ΔPA1006 aerobic cultures using Biolog plates also revealed a reduced ability to utilize a number of TCA cycle intermediates as well as a failure to utilize xanthine as a sole source of nitrogen. As a whole, these data indicate that the loss of PA1006 confers extensive changes in Pae metabolism. Based upon homology of PA1006 to the E. coli YhhP protein and data from the accompanying study, loss of PA1006 persulfuration and/or molybdenum homeostasis are likely the cause of extensive metabolic alterations that impact biofilm development and virulence in the ΔPA1006 mutant.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 6
    In: The Journal of Bacteriology, 2010, Vol. 192(9), p.2359
    Description: To fully understand how bacteria respond to their environment, it is essential to assess genome-wide transcriptional activity. New high-throughput sequencing technologies make it possible to query the transcriptome of an organism in an efficient unbiased manner. We applied a strand-specific method to sequence bacterial transcripts using Illumina's high-throughput sequencing technology. The resulting sequences were used to construct genome-wide transcriptional profiles. Novel bioinformatics analyses were developed and used in combination with proteomics data for the qualitative classification of transcriptional activity in defined regions. As expected, most transcriptional activity was consistent with predictions from the genome annotation. Importantly, we identified and confirmed transcriptional activity in areas of the genome inconsistent with the annotation and in unannotated regions. Further analyses revealed potential RpoN-dependent promoter sequences upstream of several noncoding RNAs (ncRNAs), suggesting a role for these ncRNAs in RpoN-dependent phenotypes. We were also able to validate a number of transcriptional start sites, many of which were consistent with predicted promoter motifs. Overall, our approach provides an efficient way to survey global transcriptional activity in bacteria and enables rapid discovery of specific areas in the genome that merit further investigation. doi: 10.1128/JB.01445-09
    Keywords: Promoters (Genetics) -- Research ; Bacterial Genetics -- Research ; Pseudomonas Syringae -- Genetic Aspects;
    ISSN: 0021-9193
    ISSN: 00219193
    E-ISSN: 10985530
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  • 7
    Language: English
    In: Journal of bacteriology, September 2011, Vol.193(18), pp.4598-611
    Description: The plant pathogen Pseudomonas syringae pv. tomato DC3000 (DC3000) is found in a wide variety of environments and must monitor and respond to various environmental signals such as the availability of iron, an essential element for bacterial growth. An important regulator of iron homeostasis is Fur (ferric uptake regulator), and here we present the first study of the Fur regulon in DC3000. Using chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq), 312 chromosomal regions were highly enriched by coimmunoprecipitation with a C-terminally tagged Fur protein. Integration of these data with previous microarray and global transcriptome analyses allowed us to expand the putative DC3000 Fur regulon to include genes both repressed and activated in the presence of bioavailable iron. Using nonradioactive DNase I footprinting, we confirmed Fur binding in 41 regions, including upstream of 11 iron-repressed genes and the iron-activated genes encoding two bacterioferritins (PSPTO_0653 and PSPTO_4160), a ParA protein (PSPTO_0855), and a two-component system (TCS) (PSPTO_3382 to PSPTO_3380).
    Keywords: Gene Expression Regulation, Bacterial ; Regulon ; Bacterial Proteins -- Metabolism ; Iron -- Metabolism ; Pseudomonas Syringae -- Genetics
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 8
    In: FEMS Microbiology Letters, 2017, Vol. 364(8)
    Description: The plant pathogen Pseudomonas syringae accounts for substantial crop losses and is considered an important agricultural issue. To better manage disease in the field, it is important to have an understanding of the underlying genetic mechanisms that mediate virulence. There are a substantial number of genes in sequenced bacterial genomes, including P. syringae , that encode for conserved hypothetical proteins; some of these have been functionally characterized in other Pseudomonads and have been demonstrated to play important roles in disease. PSPTO_3957 encodes a conserved hypothetical protein of unknown function. To evaluate the role of PSPTO_3957 in P. syringae pv. tomato DC3000, a PSPTO_3957 deletion mutant was constructed. Here, we show that PSPTO_3957 does not influence growth on rich media, motility or biofilm formation but is necessary for nitrate assimilation and full virulence in P. syringae . Our results have revealed an important role for PSPTO_3957 in the biology of P. syringae . Given the conservation of this protein among many bacteria, this protein might serve as an attractive target for disease management of this and other bacterial plant pathogens. Virulence of Pseudomonas syringe requires the gene PSPTO_3957.
    Keywords: 〈Kwd〉 〈Italic Toggle="Yes"〉Pseudomonas Syringae〈/Italic〉 〈/Kwd〉 ; Pa1006 ; Nitrate Assimilation ; Molybdopterin ; Virulence ; Tomato
    E-ISSN: 1574-6968
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  • 9
    Language: English
    In: PLoS ONE, 01 January 2013, Vol.8(2), p.e55593
    Description: A companion manuscript revealed that deletion of the Pseudomonas aeruginosa (Pae) PA1006 gene caused pleiotropic defects in metabolism including a loss of all nitrate reductase activities, biofilm maturation, and virulence. Herein, several complementary approaches indicate that PA1006 protein serves as a persulfide-modified protein that is critical for molybdenum homeostasis in Pae. Mutation of a highly conserved Cys22 to Ala or Ser resulted in a loss of PA1006 activity. Yeast-two-hybrid and a green-fluorescent protein fragment complementation assay (GFP-PFCA) in Pae itself revealed that PA1006 interacts with Pae PA3667/CsdA and PA3814/IscS Cys desulfurase enzymes. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) "top-down" analysis of PA1006 purified from Pae revealed that conserved Cys22 is post-translationally modified in vivo in the form a persulfide. Inductively-coupled-plasma (ICP)-MS analysis of ΔPA1006 mutant extracts revealed that the mutant cells contain significantly reduced levels of molybdenum compared to wild-type. GFP-PFCA also revealed that PA1006 interacts with several molybdenum cofactor (MoCo) biosynthesis proteins as well as nitrate reductase maturation factor NarJ and component NarH. These data indicate that a loss of PA1006 protein's persulfide sulfur and a reduced availability of molybdenum contribute to the phenotype of a ΔPA1006 mutant.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 10
    Language: English
    In: Scientific Reports, 01 July 2018, Vol.8(1), pp.1-14
    Description: Abstract Microbial biomineralization is a widespread phenomenon. The ability to induce calcium precipitation around bacterial cells has been reported in several Pseudomonas species but has not been thoroughly tested. We assayed 14 Pseudomonas strains representing five different species for the ability to precipitate calcium. Calcium phosphate precipitated adjacent to the colonies of all the Pseudomonas strains tested and also precipitated on the surface of colonies for several of the Pseudomonas strains assayed. The precipitate was commonly precipitated as amorphous calcium phosphate, however seven of the 14 Pseudomonas strains tested precipitated amorphous apatite in agar adjacent to the colonies. Out of the seven Pseudomonas strains that precipitated amorphous apatite, six are plant pathogenic. The formation of amorphous apatite was commonly observed in the area of the agar where amorphous calcium phosphate had previously formed. A transposon mutagenesis screen in Pseudomonas syringae pv. tomato DC3000 revealed genes involved in general metabolism, lipopolysaccharide and cell wall biogenesis, and in regulation of virulence play a role in calcium precipitation. These results shed light on the common ability of Pseudomonas species to perform calcium precipitation and the underlying genetic regulation involved in biomineralization.
    Keywords: Biology
    E-ISSN: 2045-2322
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