European Apis mellifera and Asian Apis cerana honeybees are essential crop pollinators. Microbiome studies can provide complex information on health and fitness of these insects in relation to environmental changes, and plant availability. Amplicon sequencing of variable regions of the 16S rRNA from bacteria and the internally transcribed spacer (ITS) regions from fungi and plants allow identification of the metabiome. These methods provide a tool for monitoring otherwise uncultured microbes isolated from the gut of the honeybees. They also help monitor the composition of the gut fungi and, intriguingly, pollen collected by the insect. Here, we present data from amplicon sequencing of the 16S rRNA from bacteria and ITS2 regions from fungi and plants derived from honeybees collected at various time points from anthropogenic landscapes such as urban areas in Poland, UK, Spain, Greece, and Thailand. We have analysed microbial content of honeybee intestine as well as fungi and pollens. Furthermore, isolated DNA was used as the template for screening pathogens: Nosema apis, N. ceranae, N. bombi, tracheal mite (Acarapis woodi), any organism in the parasitic order Trypanosomatida, including Crithidia spp. (i.e., Crithidia mellificae), neogregarines including Mattesia and Apicystis spp. (i.e., Apicistis bombi). We conclude that differences between samples were mainly influenced by the bacteria, plant pollen and fungi, respectively. Moreover, honeybees feeding on a sugar based diet were more prone to fungal pathogens (Nosema ceranae) and neogregarines. In most samples Nosema sp. and neogregarines parasitized the host bee at the same time. A higher load of fungi, and bacteria groups such as Firmicutes (Lactobacillus); γ-proteobacteria, Neisseriaceae, and other unidentified bacteria was observed for Nosema ceranae and neogregarine infected honeybees. Healthy honeybees had a higher load of plant pollen, and bacteria groups such as: Orbales, Gilliamella, Snodgrassella, and Enterobacteriaceae. Finally, the period when honeybees switch to the winter generation (longer-lived forager honeybees) is the most sensitive to diet perturbations, and hence pathogen attack, for the whole beekeeping season. It is possible that evolutionary adaptation of bees fails to benefit them in the modern anthropomorphised environment.

The transcriptional control of sporulation in Bacillus subtilis is reasonably well understood, but its translational control is underexplored. Here, we use RNA-seq, ribosome profiling and fluorescence microscopy to study the translational dynamics of B. subtilis sporulation. We identify two events of translation silencing and describe spatiotemporal changes in subcellular localization of ribosomes during sporulation. We investigate the potential regulatory role of ribosomes during sporulation using a strain lacking zinc-independent paralogs of three zinc-dependent ribosomal proteins (L31, L33 and S14). The mutant strain exhibits delayed sporulation, reduced germination efficiency, dysregulated translation of metabolic and sporulation-related genes, and disruptions in translation silencing, particularly in late sporulation.

European Apis mellifera and Asian Apis cerana honeybees, are essential crop pollinators. Microbiome studies can provide complex information on health and fitness of these insects in relation to environmental changes, and plant availability. Amplicon sequencing of variable regions of 16S rRNA and internally transcribed spacers (ITSs) allow identification of the metabiome. These methods provide a tool for monitoring otherwise uncultured microbes isolated from the gut of the honeybees. They also help monitor the composition of the gut fungi and, intriguingly, pollens collected by the insect. Here, we present data from amplicon sequencing of the 16S rRNA and ITS2 regions from honeybees collected at various time points from anthropogenic landforms as urban areas in Poland, UK, Spain, Greece, and Thailand. We have analysed microbial composition of honeybee intestine as well as fungi and pollens. We conclude that differences between samples were mainly influenced by the bacteria, plant pollens and fungi, respectively. Moreover, honeybees feeding on a honeydew diet, mainly based on sugars, were more prone to fungal pathogens ( Nosema ceranae ) and neogregarines. Finally, the period when honeybees switch to the winter generation (longer-lived forager honeybees) is the most sensitive to diet perturbations and hence pathogens attack, for the whole beekeeping season. It is possible that evolutionary adaptation of bees fails to benefit them in the modern anthropomorphised environment.

Ribosomal action is facilitated by the orchestrated work of trans -acting factors and ribosomal elements, which are subject to regulatory events, often involving phosphorylation. One such element is the ribosomal P-stalk, which plays a dual function: it activates translational GTPases, which support basic ribosomal functions, and interacts with the Gcn2 kinase, linking the ribosomes to the ISR pathway. We show that P-stalk proteins, which form a pentamer, exist in the cell exclusively in a phosphorylated state at five C-terminal domains (CTDs), ensuring optimal translation (speed and accuracy) and may play a role in the timely regulation of the Gcn2-dependent stress response. Phosphorylation of the CTD induces a structural transition from a collapsed to a coil-like structure, and the CTD gains conformational freedom, allowing specific but transient binding to various protein partners, optimizing the ribosome action. The report reveals a unique feature of the P-stalk proteins, indicating that, unlike most ribosomal proteins, which are regulated by phosphorylation in an on/off manner, the P-stalk proteins exist in a constantly phosphorylated state, which optimizes their interaction with auxiliary factors.

In the spore-forming bacterium Bacillus subtilis transcription and translation are uncoupled and the translational machinery is located at the cell poles. During sporulation, the cell undergoes morphological changes including asymmetric division and chromosome translocation into the forespore. However, the fate of translational machinery during sporulation has not been described. Here, using microscopy and mass spectrometry, we show the localisation of ribosomes during sporulation in wild type and mutant Bacillus subtilis. We demonstrate that ribosomes are associated with the asymmetric septum, a functionally important organelle in the cell’s developmental control, and that SpoIIDMP-driven peptidoglycan rearrangement is crucial for ribosomes packing into the forespore. We also show that the SpoIIIA-SpoIIQ ‘feeding-tube’ channel is not required for ribosome translocation. Our results demonstrate that translation and translational machinery are temporally and spatially organised in B. subtilis during sporulation and that the forespore ‘inherits’ ribosomes from the mother cell. We propose that the movement of ribosomes in the cell may be mediated by the bacterial homologs of cytoskeletal proteins and that the cues for asymmetric division localisation may be translation-dependent. We anticipate our findings to elicit more sophisticated structural and mechanistic studies of ribosome organisation during bacterial cell development.

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript. In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment. Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer's guidelines with some degree of optimization. The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.