Understanding the composition and function of the vaginal microbiome is crucial for reproductive and overall health. Here we established the Isala citizen-science project to analyse the vaginal microbiomes of 3,345 women in Belgium (18–98 years) through self-sampling, 16S amplicon sequencing and extensive questionnaires. The overall vaginal microbiome composition was strongly tied to age, childbirth and menstrual cycle phase.Lactobacillusspecies dominated 78% of the vaginal samples. Specific bacterial taxa also showed to co-occur in modules based on network correlation analysis. Notably, the module containingLactobacillus crispatus,Lactobacillus jenseniiandLimosilactobacillustaxa was positively linked to oestrogen levels and contraceptive use and negatively linked to childbirth and breastfeeding. Other modules, named after abundant taxa (Gardnerella,PrevotellaandBacteroides), correlated with multiple partners, menopause, menstrual hygiene and contraceptive use. With this resource-rich vaginal microbiome map and associated health, life-course, lifestyle and dietary factors, we provide unique data and insights for follow-up clinical and mechanistic research.

Mushrooms are the most conspicuous fungal structures. Transcription factors (TFs) Bri1 and Hom1 of the model fungus Schizophyllum commune are involved in late stages of mushroom development, while Wc-2, Hom2, and Fst4 function early in development. Here, it is shown that Bri1 and Hom1 also stimulate vegetative growth, while biomass formation is repressed by Wc-2, Hom2, and Fst4. The Δbri1Δbri1 and the Δhom1Δhom1 strains formed up to 0.6 fold less biomass when compared to wild-type, while Δwc-2Δwc-2, Δhom2Δhom2, and Δfst4Δfst4 strains formed up to 2.8 fold more biomass. Inactivation of TF gene tea1, which was downregulated in the Δwc-2Δwc-2, Δhom2Δhom2, and Δfst4Δfst4 strains, resulted in a strain that was severely affected in mushroom development and that produced 1.3 fold more biomass than the wild-type. In contrast, introducing a constitutive active version of hom2 that had 4 predicted phosphorylation motifs eliminated resulted in radial growth inhibition and prompt fructification in both Δhom2 and wild-type strains, even in sterile monokaryons. Together, it is concluded that TFs involved in mushroom formation also modulate vegetative growth. Among these TFs is the homeodomain protein Hom2, being the first time that this class of regulatory proteins is implicated in repression of vegetative growth in a eukaryote.

India is home to 25% of all tuberculosis cases and the second highest number of multidrug resistant cases worldwide. However, little is known about the genetic diversity and resistance determinants of Indian Mycobacterium tuberculosis, particularly for the primary lineages found in India, lineages 1 and 3. We whole genome sequenced 223 randomly selected M. tuberculosis strains from 196 patients within the Tiruvallur and Madurai districts of Tamil Nadu in Southern India. Using comparative genomics, we examined genetic diversity, transmission patterns, and evolution of resistance. Genomic analyses revealed (1) prevalence of strains from lineages 1 and 3, (2) recent transmission of strains among patients from the same treatment centers, (3) emergence of drug resistance within patients over time, (4) resistance gained in an order typical of strains from different lineages and geographies, (5) underperformance of known resistance-conferring mutations to explain phenotypic resistance in Indian strains relative to studies focused on other geographies, and (6) the possibility that resistance arose through mutations not previously implicated in resistance, or through infections with multiple strains that confound genotype-based prediction of resistance. In addition to substantially expanding the genomic perspectives of lineages 1 and 3, sequencing and analysis of M. tuberculosis whole genomes from Southern India highlight challenges of infection control and rapid diagnosis of resistant tuberculosis using current technologies. Further studies are needed to fully explore the complement of diversity and resistance determinants within endemic M. tuberculosis populations.

Many fungi are polykaryotic, containing multiple nuclei per cell. In the case of heterokaryons, there are different nuclear types within a single cell. It is unknown what the different nuclear types contribute in terms of mRNA expression levels in fungal heterokaryons. Each cell of the mushroom Agaricus bisporus contains two to 25 nuclei of two nuclear types originating from two parental strains. Using RNA-sequencing data, we assess the differential mRNA contribution of individual nuclear types and its functional impact. We studied differential expression between genes of the two nuclear types, P1 and P2, throughout mushroom development in various tissue types. P1 and P2 produced specific mRNA profiles that changed through mushroom development. Differential regulation occurred at the gene level, rather than at the locus, chromosomal, or nuclear level. P1 dominated mRNA production throughout development, and P2 showed more differentially up-regulated genes in important functional groups. In the vegetative mycelium, P2 up-regulated almost threefold more metabolism genes and carbohydrate active enzymes (cazymes) than P1, suggesting phenotypic differences in growth. We identified widespread transcriptomic variation between the nuclear types of A. bisporus. Our method enables studying nucleus-specific expression, which likely influences the phenotype of a fungus in a polykaryotic stage. Our findings have a wider impact to better understand gene regulation in fungi in a heterokaryotic state. This work provides insight into the transcriptomic variation introduced by genomic nuclear separation. Significance Fungi are a broad class of organisms that play crucial roles in a wide variety of natural and industrial processes. Some are also harmful, destroying crops or infecting immunocompromised patients. Many fungi, at some point during their life cycle, contain two different nuclei, each with different genetic content. We examine the regulation of genes from these nuclei in a mushroom-forming fungus. We find that these nuclei contribute differently to the regulation of the fungal cells, and may therefore have a different impact on their environment. Furthermore, these differences change throughout the development of different tissues. This work contributes to our understanding of fungal physiology by examining this process.

The Cys2His2 zinc finger protein gene c2h2 of Schizophyllum commune is involved in mushroom formation. Its inactivation results in a strain that is arrested at the stage of aggregate formation. In this study, the c2h2 orthologue of Agaricus bisporus was over-expressed in this white button mushroom forming basidiomycete using Agrobacterium-mediated transformation. Morphology, cap expansion rate, and total number and biomass of mushrooms were not affected by over-expression of c2h2. However, yield per day of the c2h2 over-expression strains peaked 1 day earlier. These data and expression analysis indicate that C2H2 impacts timing of mushroom formation at an early stage of development, making its encoding gene a target for breeding of commercial mushroom strains.

Recent genome-wide studies have demonstrated that fungi possess the machinery to alternatively splice pre-mRNA. However, there has not been a systematic categorization of the functional impact of alternative splicing in a fungus. We investigate alternative splicing and its functional consequences in the model mushroom forming fungus Schizophyllum commune. Alternative splicing was demonstrated for 2,285 out of 12,988 expressed genes, resulting in 20% additional transcripts. Intron retentions were the most common alternative splicing events, accounting for 33% of all splicing events, and 43% of the events in coding regions. On the other hand, exon skipping events were rare in coding regions (1%) but enriched in UTRs where they accounted for 57% of the events. Specific functional groups, including transcription factors, contained alternatively spliced genes. Alternatively spliced transcripts were regulated differently throughout development in 19% of the 2,285 alternatively spliced genes. Notably, 69% of alternatively spliced genes have predicted alternative functionality by loss or gain of functional domains, or by acquiring alternative subcellular locations. S. commune exhibits more alternative splicing than any other studied fungus. Taken together, alternative splicing increases the complexity of the S. commune proteome considerably and provides it with a rich repertoire of alternative functionality that is exploited dynamically.