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FinO-domain proteins are a widespread family of bacterial RNA-binding proteins with regulatory functions. Their target spectrum ranges from a single RNA pair, in the case of plasmid-encoded FinO, to global RNA regulons, as with enterobacterial ProQ. To assess whether the FinO domain itself is intrinsically selective or promiscuous, we determine in vivo targets of Neisseria meningitidis, which consists of solely a FinO domain. UV-CLIP-seq identifies associations with 16 small non-coding sRNAs and 166 mRNAs. Meningococcal ProQ predominantly binds to highly structured regions and generally acts to stabilize its RNA targets. Loss of ProQ alters transcript levels of >250 genes, demonstrating that this minimal ProQ protein impacts gene expression globally. Phenotypic analyses indicate that ProQ promotes oxidative stress resistance and DNA damage repair. We conclude that FinO domain proteins recognize some abundant type of RNA shape and evolve RNA binding selectivity through acquisition of additional regions that constrain target recognition. FinO-domain proteins are bacterial RNA-binding proteins with a wide range of target specificities. Here, the authors employ UV CLIP-seq and show that minimal ProQ protein of Neisseria meningitidis binds to various small non-coding RNAs and mRNAs involved in virulence.
Salmonella Typhimurium (S.Tm) is a common cause of self-limiting diarrhea. The mucosal inflammation is thought to arise from a standoff between the pathogen's virulence factors and the host's mucosal innate immune defenses, particularly the mucosal NAIP/NLRC4 inflammasome. However, it had remained unclear how this switches the gut from homeostasis to inflammation. This was studied using the streptomycin mouse model. S.Tm infections in knockout mice, cytokine inhibition and –injection experiments revealed that caspase-1 (not -11) dependent IL-18 is pivotal for inducing acute inflammation. IL-18 boosted NK cell chemoattractants and enhanced the NK cells' migratory capacity, thus promoting mucosal accumulation of mature, activated NK cells. NK cell depletion and Prf\(^{-/-}\) ablation (but not granulocyte-depletion or T-cell deficiency) delayed tissue inflammation. Our data suggest an NK cell perforin response as one limiting factor in mounting gut mucosal inflammation. Thus, IL-18-elicited NK cell perforin responses seem to be critical for coordinating mucosal inflammation during early infection, when S.Tm strongly relies on virulence factors detectable by the inflammasome. This may have broad relevance for mucosal defense against microbial pathogens.
Enterococcus faecalis and Enterococcus faecium are major nosocomial pathogens. Despite their relevance to public health and their role in the development of bacterial antibiotic resistance, relatively little is known about gene regulation in these species. RNA–protein complexes serve crucial functions in all cellular processes associated with gene expression, including post-transcriptional control mediated by small regulatory RNAs (sRNAs). Here, we present a new resource for the study of enterococcal RNA biology, employing the Grad-seq technique to comprehensively predict complexes formed by RNA and proteins in E. faecalis V583 and E. faecium AUS0004. Analysis of the generated global RNA and protein sedimentation profiles led to the identification of RNA–protein complexes and putative novel sRNAs. Validating our data sets, we observe well-established cellular RNA–protein complexes such as the 6S RNA–RNA polymerase complex, suggesting that 6S RNA-mediated global control of transcription is conserved in enterococci. Focusing on the largely uncharacterized RNA-binding protein KhpB, we use the RIP-seq technique to predict that KhpB interacts with sRNAs, tRNAs, and untranslated regions of mRNAs, and might be involved in the processing of specific tRNAs. Collectively, these datasets provide departure points for in-depth studies of the cellular interactome of enterococci that should facilitate functional discovery in these and related Gram-positive species. Our data are available to the community through a user-friendly Grad-seq browser that allows interactive searches of the sedimentation profiles (https://resources.helmholtz-hiri.de/gradseqef/).
Transcriptome analysis of individual cells by single-cell RNA-seq (scRNA-seq) has become routine for eukaryotic tissues, even being applied to whole multicellular organisms. In contrast, developing methods to read the transcriptome of single bacterial cells has proven more challenging, despite a general perception of bacteria as much simpler than eukaryotes. Bacterial cells are harder to lyse, their RNA content is about two orders of magnitude lower than that of eukaryotic cells, and bacterial mRNAs are less stable than their eukaryotic counterparts. Most importantly, bacterial transcripts lack functional poly(A) tails, precluding simple adaptation of popular standard eukaryotic scRNA-seq protocols that come with the double advantage of specific mRNA amplification and concomitant depletion of rRNA. However, thanks to very recent breakthroughs in methodology, bacterial scRNA-seq is now feasible. This short review will discuss recently published bacterial scRNA-seq approaches (MATQ-seq, microSPLiT, and PETRI-seq) and a spatial transcriptomics approach based on multiplexed in situ hybridization (par-seqFISH). Together, these novel approaches will not only enable a new understanding of cell-to-cell variation in bacterial gene expression, they also promise a new microbiology by enabling high-resolution profiling of gene activity in complex microbial consortia such as the microbiome or pathogens as they invade, replicate, and persist in host tissue.
To understand the gene regulation of an organism of interest, a comprehensive genome annotation is essential. While some features, such as coding sequences, can be computationally predicted with high accuracy based purely on the genomic sequence, others, such as promoter elements or noncoding RNAs, are harder to detect. RNA sequencing (RNA-seq) has proven to be an efficient method to identify these genomic features and to improve genome annotations. However, processing and integrating RNA-seq data in order to generate high-resolution annotations is challenging, time consuming, and requires numerous steps. We have constructed a powerful and modular tool called ANNOgesic that provides the required analyses and simplifies RNA-seq-based bacterial and archaeal genome annotation. It can integrate data from conventional RNA-seq and differential RNA-seq and predicts and annotates numerous features, including small noncoding RNAs, with high precision. The software is available under an open source license (ISCL) at https://pypi.org/project/ANNOgesic/.
The enrichment of deadwood is essential for the conservation of saproxylic biodiversity in managed forests. However, existing strategies focus on a cost‐intensive increase of deadwood amount, while largely neglecting increasing deadwood diversity.
Deadwood objects, that is logs and branches, from six tree species were experimentally sun exposed, canopy shaded and artificially shaded for 4 years, after which the alpha‐, beta‐ and gamma‐diversity of saproxylic beetles, wood‐inhabiting fungi and spiders were analysed. Analyses of beta‐diversity included the spatial distance between exposed deadwood objects. A random‐drawing procedure was used to identify the combination of tree species and sun exposure that yielded the highest gamma‐diversity at a minimum of exposed deadwood amount.
In sun‐exposed plots, species numbers in logs were higher than in shaded plots for all taxa, while in branches we observed the opposite for saproxylic beetles. Tree species affected the species numbers only of saproxylic beetles and wood‐inhabiting fungi. The beta‐diversity of saproxylic beetles and wood‐inhabiting fungi among logs was influenced by sun exposure and tree species, but beta‐diversity of spiders by sun exposure only. For all saproxylic taxa recorded in logs, differences between communities increased with increasing spatial distance.
A combination of canopy‐shaded Carpinus logs and sun‐exposed Populus logs resulted in the highest species numbers of all investigated saproxylic taxa among all possible combinations of tree species and sun‐exposure treatments.
Synthesis and applications. We recommend incorporating the enrichment of different tree species and particularly the variation in sun exposure into existing strategies of deadwood enrichment. Based on the results of our study, we suggest to combine the logs of softwood broadleaf tree species (e.g. Carpinus, Populus), hardwood broadleaf tree species (e.g. Quercus) and coniferous tree species (e.g. Pinus) under different conditions of sun exposure and distribute them spatially in a landscape to maximize the beneficial effects on overall diversity.
Primary determinants of communities in deadwood vary among taxa but are regionally consistent
(2020)
The evolutionary split between gymnosperms and angiosperms has far‐reaching implications for the current communities colonizing trees. The inherent characteristics of dead wood include its role as a spatially scattered habitat of plant tissue, transient in time. Thus, local assemblages in deadwood forming a food web in a necrobiome should be affected not only by dispersal ability but also by host tree identity, the decay stage and local abiotic conditions. However, experiments simultaneously manipulating these potential community drivers in deadwood are lacking. To disentangle the importance of spatial distance and microclimate, as well as host identity and decay stage as drivers of local assemblages, we conducted two consecutive experiments, a 2‐tree species and 6‐tree species experiment with 80 and 72 tree logs, respectively, located in canopy openings and under closed canopies of a montane and a lowland forest. We sampled saproxylic beetles, spiders, fungi and bacterial assemblages from logs. Variation partitioning for community metrics based on a unified framework of Hill numbers showed consistent results for both studies: host identity was most important for sporocarp‐detected fungal assemblages, decay stage and host tree for DNA‐detected fungal assemblages, microclimate and decay stage for beetles and spiders and decay stage for bacteria. Spatial distance was of minor importance for most taxa but showed the strongest effects for arthropods. The contrasting patterns among the taxa highlight the need for multi‐taxon analyses in identifying the importance of abiotic and biotic drivers of community composition. Moreover, the consistent finding of microclimate as the primary driver for saproxylic beetles compared to host identity shows, for the first time that existing evolutionary host adaptions can be outcompeted by local climate conditions in deadwood.
The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and posttranscriptional regulation through the RNA-binding proteins Hfq, RsmA, and RsmN have been studied in detail, global information about stable RNA-protein complexes in this human pathogen is currently lacking. Here, we implement gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73% of all transcripts and ∼40% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa, including 6S RNA with RNA polymerase and associated product RNAs (pRNAs). We observe specific clusters of noncoding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain-containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased cosedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded noncoding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell.
IMPORTANCE Stable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation and physiology in any bacterium of interest. It is therefore crucial to globally determine these complexes in order to identify and characterize new molecular players and regulation mechanisms. Pseudomonads harbor some of the largest genomes known in bacteria, encoding ∼5,500 different proteins. Here, we provide a first glimpse on which proteins and cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa. We additionally performed this analysis with bacteria subjected to the important and frequently encountered biological stress of a bacteriophage infection. We identified several molecules with established roles in a variety of cellular pathways, which were affected by the phage and can now be explored for their role during phage infection. Most importantly, we observed strong colocalization of phage transcripts and host ribosomes, indicating the existence of specialized translation mechanisms during phage infection. All data are publicly available in an interactive and easy to use browser.
Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host1. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility2,3. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding4. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses—Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing—that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation.