Mutations at the palmitoylation site of non-structural protein nsP1 of Semliki Forest virus attenuate virus replication and cause accumulation of compensatory mutations

The replicase of Semliki Forest virus (SFV) consists of four non-structural proteins, designated nsP1–4, and is bound to cellular membranes via an amphipathic peptide and palmitoylated cysteine residues of nsP1. It was found that mutations preventing nsP1 palmitoylation also attenuated virus replication. The replacement of these cysteines by alanines, or their deletion, abolished virus viability, possibly due to disruption of interactions between nsP1 and nsP4, which is the catalytic subunit of the replicase. However, during a single infection cycle, the ability of the virus to replicate was restored due to accumulation of second-site mutations in nsP1. These mutations led to the restoration of nsP1–nsP4 interaction, but did not restore the palmitoylation of nsP1. The proteins with palmitoylation-site mutations, as well as those harbouring compensatory mutations in addition to palmitoylation-site mutations, were enzymically active and localized, at least in part, on the plasma membrane of transfected cells. Interestingly, deletion of 7 aa including the palmitoylation site of nsP1 had a relatively mild effect on virus viability and no significant impact on nsP1–nsP4 interaction. Similarly, the change of cysteine to alanine at the palmitoylation site of nsP1 of Sindbis virus had only a mild effect on virus replication. Taken together, these findings indicate that nsP1 palmitoylation as such is not the factor determining the ability to bind to cellular membranes and form a functional replicase complex. Instead, these abilities may be linked to the three-dimensional structure of nsP1 and the capability of nsP1 to interact with other components of the viral replicase complex

Mutations in the nuclear localization signal of nsP2 influencing RNA synthesis, protein expression and cytotoxicity of Semliki Forest virus

The cytotoxicity of Semliki Forest virus (SFV) infection is caused partly by the non-structural protein nsP2, an essential component of the SFV replicase complex. Due to the presence of a nuclear localization signal (NLS), nsP2 also localizes in the nucleus of infected cells. The present study analysed recombinant SFV replicons and genomes with various deletions or substitutions in the NLS, or with a proline-to-glycine mutation at position 718 of nsP2 (P718G). Deletion of one or two arginine residues from the NLS or substitution of two of the arginines with aspartic acid resulted in a virus with a temperature-sensitive phenotype, and substitution of all three arginines was lethal. Thus, most of the introduced mutations severely affected nsP2 functioning in viral replication; in addition, they inhibited the ability of SFV to induce translational shut-off and kill infected cells. SFV replicons with a P718G mutation or replacement of the NLS residues 648RRR650 with RDD were found to be the least cytotoxic. Corresponding replicons expressed non-structural proteins at normal levels, but had severely reduced genomic RNA synthesis and were virtually unable to replicate and transcribe co-electroporated helper RNA. The non-cytotoxic phenotype was maintained in SFV full-length genomes harbouring the corresponding mutations; however, during a single cycle of cell culture, these were converted to a cytotoxic phenotype, probably due to the accumulation of compensatory mutations

Microbiome-based solutions to address new and existing threats to food security, nutrition, health and agrifood systems' sustainability

In addition to challenges like climate change and biodiversity loss, the sustainability and resilience of agrifood systems worldwide are currently challenged by new threats, such as the COVID-19 pandemic and the Ukraine war. Furthermore, the resilience and sustainability of our agrifood systems need to be enhanced in ways that simultaneously increase agricultural production, decrease post-harvest food losses and food waste, protect the climate, environment and health, and preserve biodiversity. The precarious situation of agrifood systems is also illustrated by the fact that overall, around 3 billion people worldwide still do not have regular access to a healthy diet. This results in various forms of malnutrition, as well as increasing number of people suffering from overweight and obesity, and diet-related, non-communicable diseases (NCDs) around the world. Findings from microbiome research have shown that the human gut microbiome plays a key role in nutrition and diet-related diseases and thus human health. Furthermore, the microbiome of soils, plants, and animals play an equally important role in environmental health and agricultural production. Upcoming, microbiome-based solutions hold great potential for more resilient, sustainable, and productive agrifood systems and open avenues toward preventive health management. Microbiome-based solutions will also be key to make better use of natural resources and increase the resilience of agrifood systems to future emerging and already-known crises. To realize the promises of microbiome science and innovation, there is a need to invest in enhancing the role of microbiomes in agrifood systems in a holistic One Health approach and to accelerate knowledge translation and implementation.YS, KD'H, LL, HS, LO, TK, EM, AM, IS, and AS received funding from the European Union's H2020 Research and Innovation Programme under Grant No. 818116 (Microbiome Support).Peer reviewe

Microbiome definition re-visited: old concepts and new challenges

peer-reviewedAbstract The field of microbiome research has evolved rapidly over the past few decades and has become a topic of great scientific and public interest. As a result of this rapid growth in interest covering different fields, we are lacking a clear commonly agreed definition of the term “microbiome.” Moreover, a consensus on best practices in microbiome research is missing. Recently, a panel of international experts discussed the current gaps in the frame of the European-funded MicrobiomeSupport project. The meeting brought together about 40 leaders from diverse microbiome areas, while more than a hundred experts from all over the world took part in an online survey accompanying the workshop. This article excerpts the outcomes of the workshop and the corresponding online survey embedded in a short historical introduction and future outlook. We propose a definition of microbiome based on the compact, clear, and comprehensive description of the term provided by Whipps et al. in 1988, amended with a set of novel recommendations considering the latest technological developments and research findings. We clearly separate the terms microbiome and microbiota and provide a comprehensive discussion considering the composition of microbiota, the heterogeneity and dynamics of microbiomes in time and space, the stability and resilience of microbial networks, the definition of core microbiomes, and functionally relevant keystone species as well as co-evolutionary principles of microbe-host and inter-species interactions within the microbiome. These broad definitions together with the suggested unifying concepts will help to improve standardization of microbiome studies in the future, and could be the starting point for an integrated assessment of data resulting in a more rapid transfer of knowledge from basic science into practice. Furthermore, microbiome standards are important for solving new challenges associated with anthropogenic-driven changes in the field of planetary health, for which the understanding of microbiomes might play a key role. Video Abstrac

Diversity and Stability of Lactic Acid Bacteria in Rye Sourdoughs of Four Bakeries with Different Propagation Parameters.

We identified the lactic acid bacteria within rye sourdoughs and starters from four bakeries with different propagation parameters and tracked their dynamics for between 5-28 months after renewal. Evaluation of bacterial communities was performed using plating, denaturing gradient gel electrophoresis, and pyrosequencing of 16S rRNA gene amplicons. Lactobacillus amylovorus and Lactobacillus frumenti or Lactobacillus helveticus, Lactobacillus pontis and Lactobacillus panis prevailed in sourdoughs propagated at higher temperature, while ambient temperature combined with a short fermentation cycle selected for Lactobacillus sanfranciscensis, Lactobacillus pontis, and Lactobacillus zymae or Lactobacillus helveticus, Lactobacillus pontis and Lactobacillus zymae. The ratio of species in bakeries employing room-temperature propagation displayed a seasonal dependence. Introduction of different and controlled propagation parameters at one bakery (higher fermentation temperature, reduced inoculum size, and extended fermentation time) resulted in stabilization of the microbial community with an increased proportion of L. helveticus and L. pontis. Despite these new propagation parameters no new species were detected

Evolution of Bacterial Consortia in Spontaneously Started Rye Sourdoughs during Two Months of Daily Propagation

<div><p>The evolution of bacterial consortia was studied in six semi-solid rye sourdoughs during long-term backslopping at different temperatures. Each rye sourdough was started spontaneously in a laboratory (dough yield 200), propagated at either 20°C or 30°C, and renewed daily at an inoculation rate of 1∶10 for 56 days. The changes in bacterial diversity over time were followed by both DGGE coupled with partial 16S rRNA gene sequencing and pyrosequencing of bar-coded 16S rRNA gene amplicons. Four species from the genus <i>Lactobacillus (brevis, crustorum, plantarum,</i> and <i>paralimentarius)</i> were detected in different combinations in all sourdoughs after 56 propagation cycles. Facultative heterofermentative lactic acid bacteria dominated in sourdoughs fermented at 30°C, while both obligate and facultative heterofermentative LAB were found to dominate in sourdoughs fermented at 20°C. After 56 propagation cycles, <i>Kazachstania unispora</i> (formerly <i>Saccharomyces unisporus</i>) was identified as the only yeast species that dominated in sourdoughs fermented at 20°C, while different combinations of strains from four yeast species (<i>Kazachstania unispora</i>, <i>Saccharomyces cerevisiae</i>, <i>Candida krusei</i> and <i>Candida glabrata)</i> were detected in sourdoughs propagated at 30°C. The evolution of bacterial communities in sourdoughs fermented at the same temperature did not follow the same time course and changes in the composition of dominant and subdominant bacterial communities occurred even after six weeks of backslopping.</p></div

Enumeration of lactic acid bacteria in spontaneous rye sourdoughs during two months of backslopping; viable counts are given as log CFU/g obtained on SDB and MRS media.

<p>*Average values were statistically different between 20°C and 30°C fermentations according to Z-test.</p

Carbohydrate fermentation profiles of <i>Lactobacillus</i> species isolated on day 56 of sourdough backlopping.

<p>Carbohydrate fermentation profiles of <i>Lactobacillus</i> species isolated on day 56 of sourdough backlopping.</p

Ratio of species in the sourdoughs fermented at 20°C (20-I, 20-II, 20-III) or 30°C (30-I, 30-II, 30-III) after 56 backslopping cycles determined by plating on MRS and SDB media or by pyrosequencing of 16S rRNA gene amplicons.

<p>Ratio of species in the sourdoughs fermented at 20°C (20-I, 20-II, 20-III) or 30°C (30-I, 30-II, 30-III) after 56 backslopping cycles determined by plating on MRS and SDB media or by pyrosequencing of 16S rRNA gene amplicons.</p