Single-cell analysis in hypersaline brines predicts a water-activity limit of microbial anabolic activity.

Hypersaline brines provide excellent opportunities to study extreme microbial life. Here, we investigated anabolic activity in nearly 6000 individual cells from solar saltern sites with water activities (aw) ranging from 0.982 to 0.409 (seawater to extreme brine). Average anabolic activity decreased exponentially with aw, with nuanced trends evident at the single-cell level: The proportion of active cells remained high (>50%) even after NaCl saturation, and subsets of cells spiked in activity as aw decreased. Intracommunity heterogeneity in activity increased as seawater transitioned to brine, suggesting increased phenotypic heterogeneity with increased physiological stress. No microbial activity was detected in the 0.409-aw brine (an MgCl2-dominated site) despite the presence of cell-like structures. Extrapolating our data, we predict an aw limit for detectable anabolic activity of 0.540, which is beyond the currently accepted limit of life based on cell division. This work demonstrates the utility of single-cell, metabolism-based techniques for detecting active life and expands the potential habitable space on Earth and beyond.

Autochthonous carbon loading of macroalgae stimulates benthic biological nitrogen fixation rates in shallow coastal marine sediments.

Macroalgae, commonly known as seaweed, are foundational species in coastal ecosystems and contribute significantly to coastal primary production globally. However, the impact of macroalgal decomposition on benthic biological nitrogen fixation (BNF) after deposition to the seafloor remains largely unexplored. In this study, we measure BNF rates at three different sites at the Big Fisherman's Cove on Santa Catalina Island, CA, USA, which is representative of globally distributed rocky bottom macroalgal habitats. Unamended BNF rates varied among sites (0.001-0.05 nmol N g-1 h -1) and were generally within the lower end of previously reported ranges. We hypothesized that the differences in BNF between sites were linked to the availability of organic matter. Indeed, additions of glucose, a labile carbon source, resulted in 2-3 orders of magnitude stimulation of BNF rates in bottle incubations of sediment from all sites. To assess the impact of complex, autochthonous organic matter, we simulated macroalgal deposition and remineralization with additions of brown (i.e., Macrocystis pyrifera and Dictyopteris), green (i.e., Codium fragile), and red (i.e., Asparagopsis taxiformis) macroalgae. While brown and green macroalgal amendments resulted in 53- to 520-fold stimulation of BNF rates-comparable to the labile carbon addition-red alga was found to significantly inhibit BNF rates. Finally, we employed nifH sequencing to characterize the diazotrophic community associated with macroalgal decomposition. We observed a distinct community shift in potential diazotrophs from primarily Gammaproteobacteria in the early stages of remineralization to a community dominated by Deltaproteobacteria (e.g., sulfate reducers), Bacteroidia, and Spirochaeta toward the latter phase of decomposition of brown, green, and red macroalgae. Notably, the nifH-containing community associated with red macroalgal detritus was distinct from that of brown and green macroalgae. Our study suggests coastal benthic diazotrophs are limited by organic carbon and demonstrates a significant and phylum-specific effect of macroalgal loading on benthic microbial communities.

Mucolipidosis types II and III and non-syndromic stuttering are associated with different variants in the same genes.

Homozygous mutations in GNPTAB and GNPTG are classically associated with mucolipidosis II (ML II) alpha/beta and mucolipidosis III (ML III) alpha/beta/gamma, which are rare lysosomal storage disorders characterized by multiple pathologies. Recently, variants in GNPTAB, GNPTG, and the functionally related NAGPA gene have been associated with non-syndromic persistent stuttering. In a worldwide sample of 1013 unrelated individuals with non-syndromic persistent stuttering we found 164 individuals who carried a rare non-synonymous coding variant in one of these three genes. We compared the frequency of these variants with those in population-matched controls and genomic databases, and their location with those reported in mucolipidosis. Stuttering subjects displayed an excess of non-synonymous coding variants compared to controls and individuals in the 1000 Genomes and Exome Sequencing Project databases. We identified a total of 81 different variants in our stuttering cases. Virtually all of these were missense substitutions, only one of which has been previously reported in mucolipidosis, a disease frequently associated with complete loss-of-function mutations. We hypothesize that rare non-synonymous coding variants in GNPTAB, GNPTG, and NAGPA may account for as much as 16% of persistent stuttering cases, and that variants in GNPTAB and GNPTG are at different sites and may in general, cause less severe effects on protein function than those in ML II alpha/beta and ML III alpha/beta/gamma.

Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking, and Persistent Stuttering.

Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-ß4-µ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the µ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering.

A study of the role of the FOXP2 and CNTNAP2 genes in persistent developmental stuttering.

A number of speech disorders including stuttering have been shown to have important genetic contributions, as indicated by high heritability estimates from twin and other studies. We studied the potential contribution to stuttering from variants in the FOXP2 gene, which have previously been associated with developmental verbal dyspraxia, and from variants in the CNTNAP2 gene, which have been associated with specific language impairment (SLI). DNA sequence analysis of these two genes in a group of 602 unrelated cases, all with familial persistent developmental stuttering, revealed no excess of potentially deleterious coding sequence variants in the cases compared to a matched group of 487 well characterized neurologically normal controls. This was compared to the distribution of variants in the GNPTAB, GNPTG, and NAGPA genes which have previously been associated with persistent stuttering. Using an expanded subject data set, we again found that NAGPA showed significantly different mutation frequencies in North Americans of European descent (p=0.0091) and a significant difference existed in the mutation frequency of GNPTAB in Brazilians (p=0.00050). No significant differences in mutation frequency in the FOXP2 and CNTNAP2 genes were observed between cases and controls. To examine the pattern of expression of these five genes in the human brain, real time quantitative reverse transcription PCR was performed on RNA purified from 27 different human brain regions. The expression patterns of FOXP2 and CNTNAP2 were generally different from those of GNPTAB, GNPTG and NAPGA in terms of relatively lower expression in the cerebellum. This study provides an improved estimate of the contribution of mutations in GNPTAB, GNPTG and NAGPA to persistent stuttering, and suggests that variants in FOXP2 and CNTNAP2 are not involved in the genesis of familial persistent stuttering. This, together with the different brain expression patterns of GNPTAB, GNPTG, and NAGPA compared to that of FOXP2 and CNTNAP2, suggests that the genetic neuropathological origins of stuttering differ from those of verbal dyspraxia and SLI.