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DNA packaging bias and differential expression of gene transfer agent genes within a population during production and release of the Rhodobacter capsulatus gene transfer agent, RcGTA

ABSTRACT: Rhodobacter capsulatus produces a gene transfer agent (GTA) called RcGTA. RcGTA is a phage-like particle that packages R. capsulatus DNA and transfers it to other R. capsulatus cells. Low-resolution techniques suggested RcGTA packages random DNA. Analysis of the RcGTA terminase sequence revealed a distant relationship to the phage T4 terminase protein, which can also perform sequence-independent packaging. We quantified the relative frequency of packaging for each gene in the genome by hybridization of DNA from RcGTA particles to an R. capsulatus microarray. All genes were found within the RcGTA particles, and random packaging was observed at a genome-wide scale. However, the genes encoding the RcGTA particle were under-packaged compared to other regions. Single-cell expression analysis demonstrated that RcGTA gene expression is not uniform within a culture. We propose a mechanism by which high levels of RcGTA gene transcription in the most active RcGTA producing cells hinders access of the packaging machinery to these genes, which causes a reduction in their packaging frequency. This sub-population gene expression phenomenon likely explains the lack of observed cell lysis in RcGTA producing cultures.

ORGANISM(S): Rhodobacter capsulatus

PROVIDER: GSE33176 | GEO | 2012/05/22

SECONDARY ACCESSION(S): PRJNA149227

REPOSITORIES: GEO

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DNA packaging bias and differential expression of gene transfer agent genes within a population during production and release of the Rhodobacter capsulatus gene transfer agent, RcGTA

Project description:Rhodobacter capsulatus produces a gene transfer agent (GTA) called RcGTA. RcGTA is a phage-like particle that packages R. capsulatus DNA and transfers it to other R. capsulatus cells. Low-resolution techniques suggested RcGTA packages random DNA. Analysis of the RcGTA terminase sequence revealed a distant relationship to the phage T4 terminase protein, which can also perform sequence-independent packaging. We quantified the relative frequency of packaging for each gene in the genome by hybridization of DNA from RcGTA particles to an R. capsulatus microarray. All genes were found within the RcGTA particles, and random packaging was observed at a genome-wide scale. However, the genes encoding the RcGTA particle were under-packaged compared to other regions. Single-cell expression analysis demonstrated that RcGTA gene expression is not uniform within a culture. We propose a mechanism by which high levels of RcGTA gene transcription in the most active RcGTA producing cells hinders access of the packaging machinery to these genes, which causes a reduction in their packaging frequency. This sub-population gene expression phenomenon likely explains the lack of observed cell lysis in RcGTA producing cultures. DNA extracted from within RcGTA particles produced by DE442, an RcGTA overproducer, was hybridized to an Affymetrix genechip custom array in order to quantify the relative frequency with which any given gene was packaged within the particles. These data were MAS5 normalized. An underlying packaging bias was suspected to be transcription related, so these frequencies were compared to expression levels within the population, as determined by hybridization of late-stationary RNA samples of DE442 and of the wildtype (SB1003) to the same custom array. These data were RMA normalized.

2012-05-21 | E-GEOD-33176 | biostudies-arrayexpress

The GtaR quorum-sensing protein negatively regulates a capsular polysaccharide gene transfer agent (RcGTA) receptor on Rhodobacter capsulatus cells

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2013-09-19 | GSE41014 | GEO

The GtaR quorum-sensing protein negatively regulates a capsular polysaccharide gene transfer agent (RcGTA) receptor on Rhodobacter capsulatus cells

2013-09-19 | E-GEOD-41014 | biostudies-arrayexpress

Comparison of the expression profiles of wild-type Rhodobacter capsulatus and a GTA hyperproducer (DE442) by RNAseq.

Project description:Gene Transfer Agents (GTAs) are small virus-like particles that transfer random fragments of host bacterial DNA between cells. Unlike true viruses, whose primary aim is self-preservation, GTAs have no preference for the spread of their own genes. GTAs are typically produced from less than 1% of the population, making in-depth analysis of producer cells problematic. Here, we compared the transcriptome of wild-type R. capsulatus and a GTA hyperproducer, DE442, in which up to 40% of cells express GTAs.

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Project description:Packaging of segmented, double-stranded RNA viral genomes requires coordination of multiple viral proteins and RNA segments. For mammalian orthoreovirus (reovirus), evidence suggests either all ten or zero viral RNA segments are simultaneously packaged in a highly coordinated process hypothesized to exclude host RNA. Accordingly, reovirus generates genome-containing virions and “genomeless” top component particles. However, despite ostensibly lacking the genome, top component particles maintain a low level of infectivity. Whether reovirus particles can package host RNA is unknown. To gain insight into reovirus packaging potential and mechanisms, we employed next-generation RNA-sequencing to define the viral and host RNA content of purified reovirus virions and top component particles. Reovirus top component particles contained double-stranded viral RNA segments in similar proportions but at reduced levels compared to virions. Top component particles also were enriched for numerous host RNAs, especially short, non-polyadenylated transcripts, that differed by reovirus strain, independent of the viral polymerase. In contrast, virions were enriched for very few host RNAs. Collectively, these findings indicate that genome packaging into reovirus virions is exquisitely selective, while incorporation of host RNAs into top component particles is more promiscuous or differentially selective and may contribute to or result from inefficient viral RNA packaging.

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Macrophage response to silica nanoparticles

Project description:Using a macrophage cell line, we demonstrate the ability of amorphous silica particles to stimulate inflammatory protein secretion and induce cytotoxicity. Whole genome microarray analysis of early gene expression changes induced by 10nm and 500nm particles showed that the magnitude of change for the majority of genes correlated more tightly with particle surface area than either particle mass or number. Gene expression changes that were size-specific were also identified, however the overall biological processes represented by all gene expression changes were nearly identical, irrespective of particle diameter. Our results suggest that on an equivalent nominal surface area basis, common biological modes of action are expected for nano- and supranano-sized silica particles. Keywords: Dose-response study

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Control of a gene transfer agent cluster in Caulobacter crescentus by transcriptional activation and anti-termination

Project description:Gene transfer agents (GTA) are domesticated prophages that cannot self-multiply and be infectious but might have been co-opted to perform biological functions for the host bacteria. Recently, Caulobacter crescentus, a bacterium best known as a model organism to study bacterial cell biology and cell cycle regulation1, has been demonstrated to produce bona fide GTA particles (CcGTA). Two direct activators of the CcGTA biosynthetic gene cluster, GafY and GafZ, have been identified, however, it is unknown how GafYZ controls transcription mechanistically or how they coordinate gene expression of the CcGTA gene cluster with other accessory genes elsewhere on the genome for an ultimate CcGTA synthesis. Here, we show that the CcGTA gene cluster is transcriptionally co-activated by GafY and an integration host factor (IHF), and by a GafZ-mediated transcription anti-termination. We present evidence that GafZ is a novel transcription anti-terminator that likely forms an anti-termination complex with RNA polymerase, NusA, NusG, and NusE to bypass transcription terminators within the 14kb CcGTA cluster. Overall, we reveal a two-tier regulation (by transcription activation and anti-termination) that coordinates the synthesis of GTA particles in C. crescentus.

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Pathways identified by toxicogenomics analysis reveal the size and dose independency of silica particles-induced toxicity in mice.

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