The development of reliable, mixed-culture biotechnological processes hinges on understanding how microbial ecosystems respond to disturbances. Here we reveal extensive phenotypic plasticity and niche complementarity in oleaginous microbial populations from a biological wastewater treatment plant. We perform meta-omics analyses (metagenomics, metatranscriptomics, metaproteomics and metabolomics) on in situ samples over 14 months at weekly intervals. Based on 1,364 de novo metagenome-assembled genomes, we uncover four distinct fundamental niche types. Throughout the time-series, we observe a major, transient shift in community structure, coinciding with substrate availability changes. Functional omics data reveals extensive variation in gene expression and substrate usage amongst community members. Ex situ bioreactor experiments confirm that responses occur within five hours of a pulse disturbance, demonstrating rapid adaptation by specific populations. Our results show that community resistance and resilience are a function of phenotypic plasticity and niche complementarity, and set the foundation for future ecological engineering efforts.

Viruses and plasmids (invasive mobile genetic elements (iMGEs)) have important roles in shaping microbial communities, but their dynamic interactions with CRISPR-based immunity remain unresolved. We analysed generation-resolved iMGE–host dynamics spanning one and a half years in a microbial consortium from a biological wastewater treatment plant using integrated meta-omics. We identified 31 bacterial metagenome-assembled genomes encoding complete CRISPR–Cas systems and their corresponding iMGEs. CRISPR-targeted plasmids outnumbered their bacteriophage counterparts by at least fivefold, highlighting the importance of CRISPR-mediated defence against plasmids. Linear modelling of our time-series data revealed that the variation in plasmid abundance over time explained more of the observed community dynamics than phages. Community-scale CRISPR-based plasmid–host and phage–host interaction networks revealed an increase in CRISPR-mediated interactions coinciding with a decrease in the dominant ‘Candidatus Microthrix parvicella’ population. Protospacers were enriched in sequences targeting genes involved in the transmission of iMGEs. Understanding the factors shaping the fitness of specific populations is necessary to devise control strategies for undesirable species and to predict or explain community-wide phenotypes.

Matching metagenomic and/or metatranscriptomic data, currently often under-used, can be useful reference for metaproteomic tandem mass spectra (MS/MS) data analysis. Here we developed a software pipeline for identification of peptides and proteins from metaproteomic MS/MS data using proteins derived from matching metagenomic (and metatranscriptomic) data as the search database, based on two novel approaches Graph2Pro (published) and Var2Pep (new). Graph2Pro retains and uses uncertainties of metagenome assembly for reference-based MS/MS data analysis. Var2Pep considers the variations found in metagenomic/metatranscriptomic sequencing reads that are not retained in the assemblies (contigs). The new software pipeline provides one stop application of both tools, and it supports the use of metagenome assembly from commonly used assemblers including MegaHit and metaSPAdes. When tested on two collections of multi-omic microbiome data sets, our pipeline significantly improved the identification rate of the metaproteomic MS/MS spectra by about two folds, comparing to conventional contig- or read-based approaches (the Var2Pep alone identified 5.6% to 24.1% more unique peptides, depending on the data set). We also showed that identified variant peptides are important for functional profiling of microbiomes. All results suggested that it is important to take into consideration of the assembly uncertainties and genomic variants to facilitate metaproteomic MS/MS data interpretation. Graphical Abstract Highlights A novel meta-proteogenomic analysis pipeline integrating Graph2Pro and Var2Pep approaches. Metaproteomic support of proteins with unknown functions. Improved functional profiling of microbiomes using variant peptides.

Staphylococcus aureus is one of the major community-acquired human pathogens, with growing multidrug-resistance, leading to a major threat of more prevalent infections to humans. A variety of virulence factors and toxic proteins are secreted during infection via the general secretory (Sec) pathway, which requires an N-terminal signal peptide to be cleaved from the N-terminus of the protein. This N-terminal signal peptide is recognized and processed by a type I signal peptidase (SPase). SPase-mediated signal peptide processing is the crucial step in the pathogenicity of S. aureus. In the present study, the SPase-mediated N-terminal protein processing and their cleavage specificity were evaluated using a combination of N-terminal amidination bottom-up and top-down proteomics-based mass spectrometry approaches. Secretory proteins were found to be cleaved by SPase, specifically and non-specifically, on both sides of the normal SPase cleavage site. The non-specific cleavages occur at the relatively smaller residues that are present next to the −1, +1, and +2 locations from the original SPase cleavage site to a lesser extent. Additional random cleavages at the middle and near the C-terminus of some protein sequences were also observed. This additional processing could be a part of some stress conditions and unknown signal peptidase mechanisms.