Biology subjects

O'Keefe, T. J.

Publications and source records attributed to O'Keefe, T. J..

4 recordsLinked to original sources

bioRxiv2024

Global niche partitioning of purine and pyrimidine cross-feeding among ocean microbes

Cross-feeding involves microbes consuming the exudates of other surrounding microbes, mediating elemental cycling. Characterizing the diversity of cross-feeding pathways in ocean microbes illuminates evolutionary forces driving self-organization of ocean ecosystems. Here, we uncover a purine and pyrimidine cross-feeding network in globally abundant groups. The cyanobacterium Prochlorococcus exudes both compound classes, which metabolic reconstructions suggest follows synchronous daily genome replication. Co-occurring heterotrophs differentiate into purine- and pyrimidine-using generalists, or specialists that use compounds for different purposes. The most abundant heterotroph, SAR11, is a specialist that uses purines as sources of energy, carbon and/or nitrogen, with subgroups differentiating along ocean-scale gradients in the supply of energy and nitrogen, in turn producing putative cryptic nitrogen cycles that link many microbes. Finally, in a SAR11 subgroup that dominates where Prochlorococcus is abundant, adenine additions to cultures inhibit DNA synthesis, poising cells for replication. We argue this subgroup uses inferred daily adenine pulses from Prochlorococcus to synchronize to the daily photosynthate supply from surrounding phytoplankton.

ecology↗

bioRxiv2022

Production and cross-feeding of nitrite within Prochlorococcus populations

Prochlorococcus is an abundant photosynthetic bacterium in the oligotrophic open ocean where nitrogen (N) often limits the growth of phytoplankton. Prochlorococcus has evolved into multiple phylogenetic clades of high-light (HL) adapted and low-light (LL) adapted cells. Within these clades, cells encode a variety of N assimilation traits that are differentially distributed among members of the population. Among these traits, nitrate (NO3-) assimilation is generally restricted to a few clades of high-light adapted cells (the HLI, HLII, and HLVI clades) and a single clade of low-light adapted cells (the LLI clade). Most, if not all, cells belonging to the LLI clade have the ability to assimilate nitrite (NO2-), with a subset of this clade capable of assimilating both NO3- and NO2-. Cells belonging to the LLI clade are maximally abundant at the top of the nitracline and near the primary NO2- maximum layer. In some ecosystems, this peak in NO2- concentration may be a consequence of incomplete assimilatory NO3- reduction by phytoplankton. This phenomenon is characterized by a bottleneck in the downstream half of the NO3- assimilation pathway and the concomitant accumulation and release of NO2- by phytoplankton cells. Given the association between LLI Prochlorococcus and the primary NO2- maximum layer, we hypothesized that some Prochlorococcus exhibit incomplete assimilatory NO3- reduction. To assess this, we monitored NO2- accumulation in batch culture for 3 Prochlorococcus strains (MIT0915, MIT0917, and SB) and 2 Synechococcus strains (WH8102 and WH7803) when grown on NO3- as the sole N source. Only MIT0917 and SB accumulated external NO2- during growth on NO3-. Approximately 20-30% of the NO3-transported into the cell by MIT0917 was released as NO2-, with the balance assimilated into biomass. We further observed that co-cultures using NO3- as the sole N source could be established for MIT0917 and a Prochlorococcus strain that can assimilate NO2- but not NO3-. In these co-cultures, the NO2- released by MIT0917 was efficiently consumed by its partner strain during balanced exponential growth. Our findings highlight the potential for emergent metabolic partnerships within Prochlorococcus populations that are mediated by the production and consumption of the N cycle intermediate, NO2-. SIGNIFICANCEEarths biogeochemical cycles are substantially driven by microorganisms and their interactions. Given that N often limits marine photosynthesis, we investigated the potential for N cross-feeding within populations of Prochlorococcus, the numerically dominant photosynthetic cell in the subtropical open ocean. During growth on NO3-, some Prochlorococcus cells release up to 30% of their N uptake as extracellular NO2-. In the wild, Prochlorococcus populations are composed of multiple functional types, including those that cannot use NO3- but can still assimilate NO2-. We show that metabolic dependencies arise when Prochlorococcus strains with complementary NO2- production and consumption phenotypes are grown together on NO3-. These findings demonstrate the potential for emergent metabolic partnerships, possibly modulating ocean nutrient gradients, that are mediated by cross-feeding of N cycle intermediates.

microbiology↗

bioRxiv2020

Coping with darkness: The adaptive response of marine picocyanobacteria to repeated light energy deprivation

The picocyanobacteria Prochlorococcus and Synechococcus are found throughout the oceans euphotic zone, where the daily light:dark cycle drives their physiology. Periodic deep mixing events can, however, move cells below this zone, depriving them of light for extended periods of time. Here we demonstrate that Prochlorococcus and Synechococcus can adapt to tolerate repeated periods of light energy deprivation. Cyanobacterial cultures kept in the dark for 3 days and then returned to the light initially required 18-26 days to resume growth, but after multiple rounds of dark exposure the strains began to regrow after only 1-2 days. This dark-tolerant phenotype was stable and heritable; cultures retained the trait across at least 18-21 generations even when grown in a standard 13:11 light:dark cycle. We found no genetic differences between the dark-tolerant and parental strains of Prochlorococcus NATL2A, indicating that an epigenetic change is responsible for the adaptation. To begin to explore this possibility, we asked whether DNA methylation - an epigenetic mechanism in bacteria - occurs in Prochlorococcus. LC-MS/MS analysis showed that while DNA methylations, including 6mA and 5mC, are found in some other Prochlorococcus strains, no methylations were detected in either the parental or dark-tolerant strain used in our experiments -i.e. the NATL2A strain. These findings suggest that Prochlorococcus utilizes a yet-to-be-determined epigenetic mechanism to adapt to the stress of extended light energy deprivation.

microbiology↗

bioRxiv2020

Mutational profiling of micro-dissected pre-malignant lesions from archived specimens

BackgroundSystematic cancer screening has led to the increased detection of pre-malignant lesions (PMLs). The absence of reliable prognostic markers has led mostly to over treatment resulting in potentially unnecessary stress, or potentially insufficient treatment and avoidable progression. Importantly, most mutational profiling studies have relied on PML synchronous to invasive cancer, or performed in patients without outcome information, hence limiting their utility for biomarker discovery. The limitations in comprehensive mutational profiling of PMLs are in large part due to the significant technical and methodological challenges: most PML specimens are small, fixed in formalin and paraffin embedded (FFPE) and lack matching normal DNA. MethodsUsing test DNA from a highly degraded FFPE specimen, multiple targeted sequencing approaches were evaluated, varying DNA input amount (3-200 ng), library preparation strategy (BE: Blunt-End, SS: Single-Strand, AT: A-Tailing) and target size (whole exome vs cancer gene panel). Variants in high-input DNA from FFPE and mirrored frozen specimens were used for PML-specific variant calling training and testing, respectively. The resulting approach was applied to profile and compare multiple regions micro-dissected (mean area 5 mm2) from 3 breast ductal carcinoma in situ (DCIS). ResultsUsing low-input FFPE DNA, BE and SS libraries resulted in 4.9 and 3.7 increase over AT libraries in the fraction of whole exome covered at 20x (BE:87%, SS:63%, AT:17%). Compared to high-confidence somatic mutations from frozen specimens, PML-specific variant filtering increased recall (BE:85%, SS:80%, AT:75%) and precision (BE:93%, SS:91%, AT:84%) to levels expected from sampling variation. Copy number alterations were consistent across all tested approaches and only impacted by the design of the capture probe-set. Applied to DNA extracted from 9 micro-dissected regions (8 PML, 1 normal epithelium), the approach achieved comparable performance, illustrated the data adequacy to identify candidate driver events (GATA3 mutations, ERBB2 or FGFR1 gains, TP53 loss) and measure intra-lesion genetic heterogeneity. ConclusionAlternate experimental and analytical strategies increased the accuracy of DNA sequencing from archived micro-dissected PML regions, supporting the deeper molecular characterization of early cancer lesions and achieving a critical milestone in the development of biology-informed prognostic markers and precision chemo-prevention strategies.

genomics↗