Genetic diversity affects the daily transcriptional oscillations of marine microbial populations

Marine microbial communities are genetically diverse but have robust synchronized daily transcriptional patterns at the genus level that are similar across a wide variety of oceanic regions. We developed a microarray-inspired gene-centric approach to resolve transcription of closely-related but distinct strains/ecotypes in high-throughput sequence data. Applying this approach to the existing metatranscriptomics datasets collected from two different oceanic regions, we found unique and variable patterns of transcription by individual taxa within the abundant picocyanobacteria Prochlorococcus and Synechococcus, the alpha Proteobacterium Pelagibacter and the eukaryotic picophytoplankton Ostreococcus. The results demonstrate that marine microbial taxa respond differentially to variability in space and time in the ocean. These intra-genus individual transcriptional patterns underlie whole microbial community responses, and the approach developed here facilitates deeper insights into microbial population dynamics

Temporal variability of nitrogen fixation and particulate nitrogen export at Station ALOHA

We present nearly 9 yrs (June 2005–December 2013) of measurements of upper‐ocean (0 m to 125 m) dinitrogen (N2) fixation rates, coupled with particulate nitrogen (PN) export at 150 m, from Station ALOHA (22° 45′N, 158°W) in the North Pacific Subtropical Gyre. Between June 2005 and June 2012, N2 fixation rates were measured based on adding the 15N2 tracer as a gas bubble. Beginning in August 2012, 15N2 was first dissolved into filtered seawater and the 15N2‐enriched water was subsequently added to N2 fixation incubations. Direct comparisons between methodologies revealed a robust relationship, with the addition of 15N2‐enriched seawater resulting in twofold greater depth‐integrated rates than those derived from adding a 15N2 gas bubble. Based on this relationship, we corrected the initial period of measurements, and the resulting rates of N2 fixation averaged 230 ± 136 μmol N m−2 d−1 for the full time series (n = 71). Analysis of the 15N isotopic composition of sinking PN, together with an isotope mass balance model, revealed that N2 fixation supported 26–47% of PN export during calendar years 2006–2013. The N export derived from these fractional contributions and measured N2 fixation rates ranged between 502 and 919 μmol N m−2 d−1, which are equivalent to rates of net community production (NCP) of 1.5 to 2.7 mol C m−2 yr−1, consistent with previous independent estimates of NCP at this site

Phytoplankton transcriptomic and physiological responses to fixed nitrogen in the California current system

Marine phytoplankton are responsible for approximately half of photosynthesis on Earth. However, their ability to drive ocean productivity depends on critical nutrients, especially bioavailable nitrogen (N) which is scarce over vast areas of the ocean. Phytoplankton differ in their preferences for N substrates as well as uptake efficiencies and minimal N requirements relative to other critical nutrients, including iron (Fe) and phosphorus. In this study, we used the MicroTOOLs high-resolution environmental microarray to examine transcriptomic responses of phytoplankton communities in the California Current System (CCS) transition zone to added urea, ammonium, nitrate, and also Fe in the late summer when N depletion is common. Transcript level changes of photosynthetic, carbon fixation, and nutrient stress genes indicated relief of N limitation in many strains of Prochlorococcus, Synechococcus, and eukaryotic phytoplankton. The transcriptomic responses helped explain shifts in physiological and growth responses observed later. All three phytoplankton groups had increased transcript levels of photosynthesis and/or carbon fixation genes in response to all N substrates. However, only Prochlorococcus had decreased transcript levels of N stress genes and grew substantially, specifically after urea and ammonium additions, suggesting that Prochlorococcus outcompeted other community members in these treatments. Diatom transcript levels of carbon fixation genes increased in response to Fe but not to Fe with N which might have favored phytoplankton that were co-limited by N and Fe. Moreover, transcription patterns of closely related strains indicated variability in N utilization, including nitrate utilization by some high-light adapted Prochlorococcus. Finally, up-regulation of urea transporter genes by both Prochlorococcus and Synechococcus in response to filtered deep water suggested a regulatory mechanism other than classic control via the global N regulator NtcA. This study indicated that co-existing phytoplankton strains experience distinct nutrient stresses in the transition zone of the CCS, an understudied region where oligotrophic and coastal communities naturally mix

Nitrogenase Gene Amplicons from Global Marine Surface Waters Are Dominated by Genes of Non-Cyanobacteria

Cyanobacteria are thought to be the main N2-fixing organisms (diazotrophs) in marine pelagic waters, but recent molecular analyses indicate that non-cyanobacterial diazotrophs are also present and active. Existing data are, however, restricted geographically and by limited sequencing depths. Our analysis of 79,090 nitrogenase (nifH) PCR amplicons encoding 7,468 unique proteins from surface samples (ten DNA samples and two RNA samples) collected at ten marine locations world-wide provides the first in-depth survey of a functional bacterial gene and yield insights into the composition and diversity of the nifH gene pool in marine waters. Great divergence in nifH composition was observed between sites. Cyanobacteria-like genes were most frequent among amplicons from the warmest waters, but overall the data set was dominated by nifH sequences most closely related to non-cyanobacteria. Clusters related to Alpha-, Beta-, Gamma-, and Delta-Proteobacteria were most common and showed distinct geographic distributions. Sequences related to anaerobic bacteria (nifH Cluster III) were generally rare, but preponderant in cold waters, especially in the Arctic. Although the two transcript samples were dominated by unicellular cyanobacteria, 42% of the identified non-cyanobacterial nifH clusters from the corresponding DNA samples were also detected in cDNA. The study indicates that non-cyanobacteria account for a substantial part of the nifH gene pool in marine surface waters and that these genes are at least occasionally expressed. The contribution of non-cyanobacterial diazotrophs to the global N2 fixation budget cannot be inferred from sequence data alone, but the prevalence of non-cyanobacterial nifH genes and transcripts suggest that these bacteria are ecologically significant

Agrobacterium-mediated transformation of sorghum: factors that affect transformation efficiency.

The results presented in this work support the hypothesis that Agrobacterium-mediated transformation of sorghum is feasible, analogous to what has been demonstrated for other cereals such as rice, maize, barley and wheat. The four factors that we found most influenced transformation were: the sensitivity of immature sorghum embryos to Agrobacterium infection, the growth conditions of the donor plant, type of explant and co-cultivation medium. A major problem during the development of our protocol was a necrotic response which developed in explants after co-cultivation. Immature sorghum embryos proved to be very sensitive to Agrobacterium infection and we found that the level of embryo death after co-cultivation was the limiting step in improving transformation efficiency. The addition of coconut water to the co-cultivation medium, the use of vigorous and actively growing immature embryos and the removal of excess bacteria significantly improved the survival rate of sorghum embryos and was critical for successful transformation. Hygromycin phosphotransferase (hpt) proved to be a good selectable marker for sorghum. We also found that β-glucuronidase (GUS) activity was low in most of the transgenic plant tissues tested, although it was very high in immature inflorescences. Although promising, the overall transformation efficiency of the protocol is still low and further optimization will require particular attention to be given to the number of Agrobacterium in the inoculum and the selection of sorghum genotypes and explants less sensitive to Agrobacterium infection