Use of carbon monoxide and hydrogen by a bacteria–animal symbiosis from seagrass sediments

The gutless marine worm Olavius algarvensis lives in symbiosis with chemosynthetic bacteria that provide nutrition by fixing carbon dioxide (CO₂) into biomass using reduced sulfur compounds as energy sources. A recent metaproteomic analysis of the O. algarvensis symbiosis indicated that carbon monoxide (CO) and hydrogen (H₂) might also be used as energy sources. We provide direct evidence that the O. algarvensis symbiosis consumes CO and H₂. Single cell imaging using nanoscale secondary ion mass spectrometry revealed that one of the symbionts, the γ3‐symbiont, uses the energy from CO oxidation to fix CO₂. Pore water analysis revealed considerable in‐situ concentrations of CO and H₂ in the O. algarvensis environment, Mediterranean seagrass sediments. Pore water H₂ concentrations (89–2147 nM) were up to two orders of magnitude higher than in seawater, and up to 36‐fold higher than previously known from shallow‐water marine sediments. Pore water CO concentrations (17–51 nM) were twice as high as in the overlying seawater (no literature data from other shallow‐water sediments are available for comparison). Ex‐situ incubation experiments showed that dead seagrass rhizomes produced large amounts of CO. CO production from decaying plant material could thus be a significant energy source for microbial primary production in seagrass sediments.     

Structure and dynamics of human Nedd4-1 WW3 in complex with the αENaC PY motif

Nedd4-1 (neuronal precursor cell expressed developmentally downregulated gene 4-1) is an E3 ubiquitin ligase that interacts with and negatively regulates the epithelial Na⁺ channel (ENaC). The WW domains of Nedd4-1 bind to the ENaC subunits via recognition of PY motifs. Human Nedd4-1 (hNedd4-1) contains four WW domains with the third domain (WW3*) showing the strongest affinity to the PY motif. To understand the mechanism underlying this binding affinity, we have carried out NMR structural and dynamics analyses of the hNedd4-1 WW3* domain in complex with a peptide comprising the C-terminal tail of the human ENaC α-subunit. The structure reveals that the peptide interacts in a similar manner to other WW domain–ENaC peptide structures. Crucial interactions that likely provide binding affinity are the broad XP groove facilitating additional contacts between the WW3* domain and the peptide, compared to similar complexes, and the large surface area buried (83 Å²) between R430 (WW3*) and L647′ (αENaC). This corroborates the model-free analysis of the ¹⁵N backbone relaxation data, which showed that R430 is the most rigid residue in the domain (S² = 0.90 ± 0.01). Carr–Purcell–Meiboom–Gill relaxation dispersion analysis identified two different conformational exchange processes on the μs–ms time-scale. One of these processes involves residues located at the peptide binding interface, suggesting conformational exchange may play a role in peptide recognition. Thus, both structural and dynamic features of the complex appear to define the high binding affinity. The results should aid interpretation of biochemical data and modeling interfaces between Nedd4-1 and other interacting proteins.     

Strategies for the highly efficient synthesis of erythropoietin N‐glycopeptide hydrazides

A convergent synthesis for erythropoietin (EPO) 1‐28 N‐glycopeptide hydrazides was developed. In this approach, EPO 1‐28 peptides were synthesized on the solid phase and converted to C‐terminal hydrazides after cleavage from the resin. After selective deprotection of the Asp24 side chain, the desired glycosylamine was coupled by pseudoproline‐assisted Lansbury aspartylation. Although the initial yields of the EPO 1‐28 glycopeptides were satisfactory, they could be markedly improved by increasing the purity of the peptide using a reversed‐phase high‐performance liquid chromatography (RP‐HPLC) purification of the protected peptide.     

Defective interfering viral particles in acute dengue infections

While much of the genetic variation in RNA viruses arises because of the error-prone nature of their RNA-dependent RNA polymerases, much larger changes may occur as a result of recombination. An extreme example of genetic change is found in defective interfering (DI) viral particles, where large sections of the genome of a parental virus have been deleted and the residual sub-genome fragment is replicated by complementation by co-infecting functional viruses. While most reports of DI particles have referred to studies in vitro, there is some evidence for the presence of DI particles in chronic viral infections in vivo. In this study, short fragments of dengue virus (DENV) RNA containing only key regulatory elements at the 3' and 5' ends of the genome were recovered from the sera of patients infected with any of the four DENV serotypes. Identical RNA fragments were detected in the supernatant from cultures of Aedes mosquito cells that were infected by the addition of sera from dengue patients, suggesting that the sub-genomic RNA might be transmitted between human and mosquito hosts in defective interfering (DI) viral particles. In vitro transcribed sub-genomic RNA corresponding to that detected in vivo could be packaged in virus like particles in the presence of wild type virus and transmitted for at least three passages in cell culture. DENV preparations enriched for these putative DI particles reduced the yield of wild type dengue virus following co-infections of C6-36 cells. This is the first report of DI particles in an acute arboviral infection in nature. The internal genomic deletions described here are the most extensive defects observed in DENV and may be part of a much broader disease attenuating process that is mediated by defective viruses.     

37 A comparative study of ribosomal proteins: linkage between amino acid distribution and ribosomal assembly

Assembly of the ribosome from its protein and RNA constituents has been studied extensively over the past 50?years, and here we utilize a comparative analysis approach to relate the composition of ribosomal proteins (r-proteins) to their role in the assembly process. We computed the amino acid distributions for the 30S subunit r-protein sequences from 560 bacterial species and compared this composition to those of other house-keeping proteins from the same species. We found that r-proteins have a significantly higher content of positively charged residues (Lysine, K, and Arginine, R) than do nonribosomal proteins (10% for R and 11% for K in r-proteins, vs. 4.7% R and 5.9% K in non-ribosomal proteins), which is consistent with prior knowledge of net positive charges carried by r-proteins (Baker et al., 2001; Klein et al., 2004; Burton et al., 2012). Furthermore, these two residues are also highly represented at contact sites along the protein/RNA interface (contact enrichment factor (CEF)?>?1). These results provide further evidence of the importance of electrostatic interactions between the positively charged proteins and negatively charged ribosomal RNA (rRNA) during ribosome assembly. Other highly represented contact residues include polar and aromatic residues, which are likely to interact with rRNA via hydrogen bonds and base stacking interactions, respectively. Interestingly, the proportion of K residues generally decreases with r-protein size, reflecting a negative correlation between protein lengths and the proportion of K (Spearman’s rank correlation, ρ?=??0.802, p?=?2.60e???5). We suggest that this trend helps the smaller r-proteins, which experience higher translational entropy than large proteins, overcome the increased free energy barrier during assembly. When the r-protein sequences were categorized according to the species’ optimal growth temperature, we found that thermophiles show increased R, Isoleucine (I), and Tyrosine (Y) content, whereas mesophiles have increased proportions of Serine (S) and Threonine (T). These results reflect one typical distinction between thermophiles and mesophiles (Kumar and Nussinov 2001), yet these differences in amino acid distributions do not extend to their respective contact sites. That is, the makeup of thermophilic and mesophilic r-protein contact residues are not significantly different (p?>?0.01). This indicates that, while the percent compositions of amino acids relating to qualities such as thermostability and protein folding are expected to vary with environmental temperature, the distributions of residues in contact with rRNA are comparable for all bacterial species. From this, we conclude that the electrostatic interactions that guide ribosome assembly are independent of temperature.     

Taxonomic significance of asymmetrical helmet and lance bristles in the genus Mallomonas (Synurophyceae) and their discovery in Eocene lake sediments

Complex bristle types formed by species in the genus Mallomonas include those with helmet or lance-shaped apices. The ornamentation on each side of the helmet has been thought to be equivalent or symmetrical, whereas on a lance-shaped bristle an expanded portion folds over one side of the shaft to form an asymmetrical structure. We describe, for the first time, helmet bristles with a distinctly asymmetrical design, also formed by the folding of a siliceous membrane over one side of the helmet. We postulate that the asymmetrical helmet represents a structure that combines the formation of a symmetrical helmet and a lance-shaped design on the same bristle. Further, we report structurally similar asymmetrical helmet bristles, lance-shaped bristles and scales that are unambiguously assigned to Mallomonas asmundiae in Middle Eocene sediments from a maar lake in northern Canada, supporting the hypothesis that scale and bristle morphology in the Synurophyceae has undergone extensive prolonged evolutionary stasis. Given differences in scale morphology and the presence of asymmetrical helmet bristles, we transfer the North American endemic Mallomonas acaroides var. muskokana to the rank of species. Further, we formally describe Mallomonas dispar and M. lancea, fossil species with asymmetrical helmet bristles and lance-shaped bristles, respectively. The taxonomic and biogeographic significance of asymmetrical and lance-bearing bristles is discussed.