Host lifestyle affects human microbiota on daily timescales

Background: Disturbance to human microbiota may underlie several pathologies. Yet, we lack a comprehensive understanding of how lifestyle affects the dynamics of human-associated microbial communities. Results: Here, we link over 10,000 longitudinal measurements of human wellness and action to the daily gut and salivary microbiota dynamics of two individuals over the course of one year. These time series show overall microbial communities to be stable for months. However, rare events in each subjects’ life rapidly and broadly impacted microbiota dynamics. Travel from the developed to the developing world in one subject led to a nearly two-fold increase in the Bacteroidetes to Firmicutes ratio, which reversed upon return. Enteric infection in the other subject resulted in the permanent decline of most gut bacterial taxa, which were replaced by genetically similar species. Still, even during periods of overall community stability, the dynamics of select microbial taxa could be associated with specific host behaviors. Most prominently, changes in host fiber intake positively correlated with next-day abundance changes among 15% of gut microbiota members. Conclusions: Our findings suggest that although human-associated microbial communities are generally stable, they can be quickly and profoundly altered by common human actions and experiences.National Science Foundation (U.S.) (Grant 0821391

Silencing of the Violaxanthin De-Epoxidase Gene in the Diatom Phaeodactylum tricornutum Reduces Diatoxanthin Synthesis and Non-Photochemical Quenching

Diatoms are a major group of primary producers ubiquitous in all aquatic ecosystems. To protect themselves from photooxidative damage in a fluctuating light climate potentially punctuated with regular excess light exposures, diatoms have developed several photoprotective mechanisms. The xanthophyll cycle (XC) dependent non-photochemical chlorophyll fluorescence quenching (NPQ) is one of the most important photoprotective processes that rapidly regulate photosynthesis in diatoms. NPQ depends on the conversion of diadinoxanthin (DD) into diatoxanthin (DT) by the violaxanthin de-epoxidase (VDE), also called DD de-epoxidase (DDE). To study the role of DDE in controlling NPQ, we generated transformants of P. tricornutum in which the gene (Vde/Dde) encoding for DDE was silenced. RNA interference was induced by genetic transformation of the cells with plasmids containing either short (198 bp) or long (523 bp) antisense (AS) fragments or, alternatively, with a plasmid mediating the expression of a self-complementary hairpin-like construct (inverted repeat, IR). The silencing approaches generated diatom transformants with a phenotype clearly distinguishable from wildtype (WT) cells, i.e. a lower degree as well as slower kinetics of both DD de-epoxidation and NPQ induction. Real-time PCR based quantification of Dde transcripts revealed differences in transcript levels between AS transformants and WT cells but also between AS and IR transformants, suggesting the possible presence of two different gene silencing mediating mechanisms. This was confirmed by the differential effect of the light intensity on the respective silencing efficiency of both types of transformants. The characterization of the transformants strengthened some of the specific features of the XC and NPQ and confirmed the most recent mechanistic model of the DT/NPQ relationship in diatoms

Relative quantification of <i>Dde</i> transcripts in the WT and five <i>Dde</i> transformants of <i>Phaeodactylum tricornutum</i>.

<p>Transcript levels are relative to the WT and normalized to <i>Gapdh</i> expression. Values are averages of at least two replicates. Black bars: total RNA, untreated RNA used for qPCR; Grey bars: ssRNA, single stranded RNA (total RNA treated with RNaseIII).</p

NPQ development in WT and the <i>Dde</i> transformants of <i>Phaeodactylum tricornutum</i>.

<p>Cells were grown at low light (45 µmol photons⋅m⁻²⋅s⁻¹) before the experiment. (A) and (B) show the respective NPQs as a function of light intensity for a 5 min light exposure, while in (C) and (D) NPQ was measured as a function of time at an irradiance of 450 µmol photons⋅m⁻²⋅s⁻¹. Values are averages ± standard deviation (SD) of three to four measurements.</p

Diadinoxanthin and diatoxanthin (DD and DT) content and de-epoxidation state (DES) of the WT and <i>Dde</i> transformants of <i>Phaeodactylum tricornutum</i> grown under low light (LL).

<p>Diadinoxanthin (DD) and diatoxanthin (DT) content (in mol/100 mol Chl <i>a</i>), and the de-epoxidation state (DES) of the WT (+/− dithiothreitol, DTT) and the <i>Dde</i> transformants of <i>P. tricornutum</i> cells (LL grown) after a 5 min 450 µmol photons⋅m⁻²⋅s⁻¹ light treatment. DES (in%)  =  DT/(DD+DT) × 100. Values are averages ± SD of three to five measurements.</p

De-epoxidation state (DES) and the relationship between the increase of the diadinoxanthin+diatoxanthin (DD+DT) and the diatoxanthin (DT) amounts in WT cells and the <i>Dde</i> transformants of <i>Phaeodactylum tricornutum</i>.

<p>(A): De-epoxidation state (DES) of the WT and the <i>Dde</i> transformants of <i>P. tricornutum</i> cells grown under low (LL) and ‘high’ (HL) light (45 and 135 µmol photons⋅m⁻²⋅s⁻¹, black and white column, respectively) after a 5 min 450 µmol photons⋅m⁻²⋅s⁻¹ light treatment. For the HL cells, the DES obtained during the 450 µmol photons⋅m⁻²⋅s⁻¹ light treatment was calculated as the total DES (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036806#pone-0036806-t003" target="_blank">table 3</a>, data ‘After LT’) minus the DES which already occurred before the light treatment during the HL acclimation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036806#pone-0036806-t003" target="_blank">table 3</a>, data ‘Before LT’). Such a calculation was not necessary for the LL acclimated cells as no DD de-epoxidation occurred before the light treatment (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036806#pone-0036806-t001" target="_blank">table 1</a>, no presence of DT as this low intensity). Values are average, ± SD of three to four measurements. (B): Relationship between the increase in the total DD+DT pool size and the DT amount synthesized during the light treatment in HL grown cells compared to LL grown cells. For the HL cells, the DT amount synthesized during the 450 µmol photons⋅m⁻²⋅s⁻¹ light treatment was calculated as the total DT amount (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036806#pone-0036806-t003" target="_blank">table 3</a>, data ‘After LT’) minus the DT amount which already occurred before the light treatment during the HL acclimation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036806#pone-0036806-t003" target="_blank">table 3</a>, data ‘Before LT’). Thus, the x-axis refers to the increase of the DD+DT pool size under HL acclimation versus the LL conditions while the y-axis refers to the increase of the DT synthesis of cells acclimated to HL versus the DT synthesis of cells acclimated to LL. As a consequence, ‘LL conditions’ refers to the value of the relationship for all type of cells and is equal to 1. The linear relationship obtained for the WT and the IR transformants was DT  = 1.02 (DD+DT) with R  = 0.99. All data were extracted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036806#pone-0036806-t001" target="_blank">tables 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036806#pone-0036806-t002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036806#pone-0036806-t003" target="_blank">3</a>. See the text for details.</p

Schematic vector maps of the silencing constructs used for transformation.

<p>Anti-sense constructs: <i>Dde</i> fragments of 198 or 523 bps were cloned in anti-sense orientation downstream of the <i>fcpA</i> promoter (pDDE-AS198 and pDDE-AS523). Inverted repeat constructs: <i>Dde</i> fragments of 293 and 523 bp lengths were cloned in sense and anti-sense orientation. The two fragments were linked with an <i>eGFP</i> fragment supposed to function as spacer. Amp: Ampicillin resistance; Zeo: Zeocin™ resistance; fcpA: Fucoxanthin Chlorophyll <i>a</i>/<i>c</i>-binding Protein A promoter; eGFP: enhanced green fluorescent protein.</p

Pigments and photosynthetic properties of WT and the <i>Dde</i> transformants of <i>Phaeodactylum tricornutum.</i>

<p>Pigment content (in mol/100 mol Chl <i>a</i>) and photosynthetic properties of the WT and the <i>Dde</i> transformants of <i>P. tricornutum</i> cells grown under low light (45 µmol photons⋅m⁻²⋅s⁻¹). Chl <i>a</i> is given in pg cell⁻¹, DD: diadinoxanthin, F_v/F_m: the maximum photosynthetic efficiency of photosystem (PS) II, rETR_max: is the relative maximal rate of linear electron transport (µmol photons⋅m⁻²⋅s⁻¹), α: the maximum light used efficiency (in µmol photons⋅m⁻²⋅s⁻¹), E_m: the light intensity needed to reach rETR_max in µmol photons⋅m⁻²⋅s⁻¹, μ: the growth rate in d⁻¹. Values are averages, ± SD of seven to nine measurements for the pigment data (except Chl <i>a</i>, three measurements) and three to four measurements for the other data.</p

Host lifestyle affects human microbiota on daily timescales (vol 15, R89, 2014)

As a result of a production error during the type-setting of the final version of the article [1], a number of additional files were incorrectly published, with the files not matching the Additional Files legends. All additional files for this article are republished below in the correct order. The publisher apologizes for the error and any confusion caused

Unlocking Short Read Sequencing for Metagenomics

Background Different high-throughput nucleic acid sequencing platforms are currently available but a trade-off currently exists between the cost and number of reads that can be generated versus the read length that can be achieved. Methodology/Principal Findings We describe an experimental and computational pipeline yielding millions of reads that can exceed 200 bp with quality scores approaching that of traditional Sanger sequencing. The method combines an automatable gel-less library construction step with paired-end sequencing on a short-read instrument. With appropriately sized library inserts, mate-pair sequences can overlap, and we describe the SHERA software package that joins them to form a longer composite read. Conclusions/Significance This strategy is broadly applicable to sequencing applications that benefit from low-cost high-throughput sequencing, but require longer read lengths. We demonstrate that our approach enables metagenomic analyses using the Illumina Genome Analyzer, with low error rates, and at a fraction of the cost of pyrosequencing.Gordon and Betty Moore Foundation (Marine Microbiology Initiative)Center for Microbial Oceanography: Research and EducationUnited States. Dept. of Energy (Genome-to-Life)Natural Sciences and Engineering Research Council of CanadaFonds québécois de la recherche sur la nature et les technologie