Transcriptional response of <i>Silicibacter pomeroyi</i> DSS‐3 to dimethylsulfoniopropionate (DMSP)

Bürgmann, Helmut; Howard, Erinn C.; Ye, Wenying; Sun, Feng; Sun, Sheng; Napierala, Sarah; Moran, Mary Ann

doi:10.1111/j.1462-2920.2007.01386.x

Cited by 60 publications

(79 citation statements)

References 53 publications

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“…It has been applied to both bacterial isolates and environmental samples, in one or two rounds 6,[13][14][15][16][17][18] . The Epicentre kit uses exonuclease to preferentially degrade processed RNAs with 5′ monophosphate (the majority of which are believed to be rRNAs) 19,20 . In some instances, these methods have been used in combination to improve rRNA removal [21][22][23] .…”

Section: Illumina Sequencing Itself Also Can Compromise Quantitative mentioning

confidence: 99%

Validation of two ribosomal RNA removal methods for microbial metatranscriptomics

et al. 2010

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The predominance of rRNAs in the transcriptome is a major technical challenge in sequence-based analysis of cDNAs from microbial isolates and communities. Several approaches have been applied to deplete rRNAs from (meta)transcriptomes, but no systematic investigation of potential biases introduced by any of these approaches has been reported. Here we validated the effectiveness and fidelity of the two most commonly used approaches, subtractive hybridization and exonuclease digestion as well as combinations of these treatments, on two synthetic five-microorganism metatranscriptomes using massively parallel sequencing. We found that the effectiveness of rRNA removal was a function of community composition and RNA integrity for these treatments. Subtractive hybridization alone introduced the least bias in relative transcript abundance, whereas exonuclease and in particular combined treatments greatly compromised mRNA abundance fidelity. Illumina sequencing itself also can compromise quantitative data analysis by introducing a G+C bias between runs.Rapid technological advances in ultra-high-throughput sequencing are making de novo sequencing of transcriptomes (RNA-seq) a viable alternative to microarray analysis of microbial isolates and communities 1 . A major technical challenge for de novo transcriptome sequencing is the low relative abundance of mRNAs in total cellular RNA (1-5%; ref.2), the bulk of which is rRNAs and tRNAs 3 . Unlike eukaryotic mRNAs, which can be selectively synthesized into cDNA by virtue of their poly(A) tails 4 , bacterial and archaeal cDNAs are predominantly rRNA -2-sequences 5,6 . Therefore, prokaryotic rRNAs are often removed before sequencing to improve mRNA detection sensitivity. Different methods have been used to eliminate prokaryotic rRNA, including subtractive hybridization with rRNA-specific probes 7,8 , digestion with exonuclease that preferentially acts on rRNA, poly(A) tail addition to discriminate against rRNA 9,10 , reverse transcription with rRNA-specific primers followed by RNase H digestion to degrade rRNA:DNA hybrids 11 , and gel electrophoresis size separation and extraction of non-rRNA bands 12 .Among these methods, subtractive hybridization and exonuclease digestion have become the most popular owing to the availability of commercial kits from Ambion (MICROBExpress Bacterial mRNA Enrichment kit) and Epicentre (mRNA-ONLY Prokaryotic mRNA Isolation kit). The former kit uses a subtractive hybridization with capture oligonucleotides specific to 16S and 23S rRNAs. It has been applied to both bacterial isolates and environmental samples, in one or two rounds 6,[13][14][15][16][17][18] . The Epicentre kit uses exonuclease to preferentially degrade processed RNAs with 5′ monophosphate (the majority of which are believed to be rRNAs) 19,20 . In some instances, these methods have been used in combination to improve rRNA removal [21][22][23] . There is no consensus, however, on the best approach, and existing data are anecdotal. Here we validated the performance of these kits wit...

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Section: Illumina Sequencing Itself Also Can Compromise Quantitative mentioning

confidence: 99%

Validation of two ribosomal RNA removal methods for microbial metatranscriptomics

et al. 2010

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“…A competing metabolic pathway results in the production of dimethylsulfide (DMS) from DMSP (16, 21). DMS represents a major source of biogenic sulfur to the atmosphere, where oxidation products form cloud condensation nuclei and ultimately influence radiative backscatter (2,33,49).Recent insights into the molecular mechanisms that drive bacterial DMSP degradation have provided an improved understanding of DMSP cycling at the genomic and transcriptional levels (5,18,44,54,56,57). The identification of the DMSP demethylase gene (dmdA), which encodes the first step in the demethylation pathway, has enabled quantification of the gene in marine metagenomic surveys and revealed it to be taxonomically diverse and highly abundant (present in Ͼ50% of marine bacterioplankton) (19).…”

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confidence: 99%

Bacterial Dimethylsulfoniopropionate Degradation Genes in the Oligotrophic North Pacific Subtropical Gyre

Varaljay

Gifford

Wilson

et al. 2012

Appl Environ Microbiol

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ABSTRACTDimethylsulfoniopropionate (DMSP) is an organic sulfur compound that is rapidly metabolized by marine bacteria either by cleavage to dimethylsulfide (DMS) or demethylation to 3-methiolpropionate. The abundance and diversity of genes encoding bacterial DMS production (dddP) and demethylation (dmdA) were measured in the North Pacific subtropical gyre (NPSG) between May 2008 and February 2009 at Station ALOHA (22°45′N, 158°00′W) at two depths: 25 m and the deep chlorophyll maximum (DCM; ∼100 m). The highest abundance ofdmdAgenes was in May 2008 at 25 m, with ∼16.5% of cells harboring a gene in one of the eight subclades surveyed, while the highest abundance ofdddPgenes was in July 2008 at 25 m, with ∼2% of cells harboring a gene. ThedmdAgene pool was consistently dominated by homologs from SAR11 subclades, which was supported by findings in metagenomic data sets derived from Station ALOHA. Expression of the SAR11dmdAgenes was low, with typical transcript:gene ratios between 1:350 and 1:1,400. The abundance of DMSP genes was statistically different between 25 m and the DCM and correlated with a number of environmental variables, including primary production, photosynthetically active radiation, particulate DMSP, and DMS concentrations. At 25 m,dddPabundance was positively correlated with pigments that are diagnostic of diatoms; at the DCM,dmdAabundance was positively correlated with temperature. Based on gene abundance, we hypothesize that SAR11 bacterioplankton dominate DMSP cycling in the oligotrophic NPSG, with lesser but consistent involvement of other members of the bacterioplankton community.

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“…Whole genome microarrays have thus far been applied for marine cyanobacteria Synechococcus spp. (Palenik et al, 2003(Palenik et al, , 2006Su et al, 2006) and recently a DMSP degrading Proteobacterium Silicibacter pomeroy from the Roseobacter clade (Burgmann et al, 2007). Genome data and analyses of several marine microbes have been published, including the uncultivated marine Archaeon Cenarchaeum symbiosum , the anoxygenic anaerobic phototrophic Proteobacterium Roseobacter denitrificans (Swingley et al, 2007), the cyanobacterial diazotroph Crocosphaera watsonii , and a marine planctomycete Pirellula sp.…”

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confidence: 99%

Molecular biology techniques and applications for ocean sensing

2009

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Abstract. The study of marine microorganisms using molecular biological techniques is now widespread in the ocean sciences. These techniques target nucleic acids which record the evolutionary history of microbes, and encode for processes which are active in the ocean today. Molecular techniques can form the basis of remote instrumentation sensing technologies for marine microbial diversity and ecological function. Here we review some of the most commonly used molecular biological techniques. These techniques include the polymerase chain reaction (PCR) and reversetranscriptase PCR, quantitative PCR, whole assemblage "fingerprinting" approaches (based on nucleic acid sequence or length heterogeneity), oligonucleotide microarrays, and high-throughput shotgun sequencing of whole genomes and gene transcripts, which can be used to answer biological, ecological, evolutionary and biogeochemical questions in the ocean sciences. Moreover, molecular biological approaches may be deployed on ocean sensor platforms and hold promise for tracking of organisms or processes of interest in near-real time.

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Transcriptional response of Silicibacter pomeroyi DSS‐3 to dimethylsulfoniopropionate (DMSP)

Cited by 60 publications

References 53 publications

Validation of two ribosomal RNA removal methods for microbial metatranscriptomics

Validation of two ribosomal RNA removal methods for microbial metatranscriptomics

Bacterial Dimethylsulfoniopropionate Degradation Genes in the Oligotrophic North Pacific Subtropical Gyre

Molecular biology techniques and applications for ocean sensing

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