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Social bees harbor conserved gut microbiota that may have been acquired in a common ancestor of social bees and subsequently co-diversified with their hosts. However, most of this knowledge is based on studies on the gut microbiota of honey bees and bumble bees. Much less is known about the gut microbiota of the third and most diverse group of social bees, the stingless bees. Specifically, the absence of genomic data from their microbiota presents an important knowledge gap in understanding the evolution and functional diversity of the social bee microbiota. Here we combined community profiling with culturing and genome sequencing of gut bacteria from six neotropical stingless bee species from Brazil. Phylogenomic analyses show that most stingless bee gut isolates form deep-branching sister clades of core members of the honey bee and bumble bee gut microbiota with conserved functional capabilities, confirming the common ancestry and ecology of their microbiota. However, our bacterial phylogenies were not congruent with those of the host indicating that the evolution of the social bee gut microbiota was not driven by strict co-diversification, but included host switches and independent symbiont gain and losses. Finally, as reported for the honey bee and bumble bee microbiota, we find substantial genomic divergence among strains of stingless bee gut bacteria suggesting adaptation to different host species and glycan niches. Our study offers first insights into the genomic diversity of the stingless bee microbiota, and highlights the need for broader samplings to understand the evolution of the social bee gut microbiota.ImportanceStingless bees are the most diverse group of the corbiculate bees and represent important pollinator species throughout the tropics and subtropics. They harbor specialized microbial communities in their gut that are related to those found in honey bees and bumble bees and that are likely important for bee health. Few bacteria have been cultured from the gut of stingless bees which has prevented characterization of their genomic diversity and functional potential. Here, we established cultures of major community members of the gut microbiota of six stingless bee species and sequenced their genomes. We find that most stingless bee isolates belong to novel bacterial species distantly related to those found in honey bees and bumble bees and encoding similar functional capabilities. Our study offers a new perspective on the evolution of the social bee gut microbiota and presents the basis to characterize the symbiotic relationships between gut bacteria and stingless bees.

Antibiotic resistance in the important opportunistic human pathogen Streptococcus pneumoniae is on the rise. This is particularly problematic in the case of the β-lactam antibiotic amoxicillin, which is the first-line therapy. It is therefore crucial to uncover targets that would kill or resensitize amoxicillin-resistant pneumococci. To do so, we developed a genome-wide, single-cell based, gene silencing screen using CRISPR interference called sCRilecs-seq (subsets of CRISPR interference libraries extracted by fluorescence activated cell sorting coupled to next generation sequencing). Since amoxicillin affects growth and division, sCRilecs-seq was used to identify targets that are responsible for maintaining proper cell size. Our screen revealed that downregulation of the mevalonate pathway leads to extensive cell elongation. Further investigation into this phenotype indicates that it is caused by insufficient transport of cell wall precursors across the cell membrane due to a limitation in the production of undecaprenyl phosphate (Und-P), the lipid carrier responsible for this process. The data suggest that whereas peptidoglycan synthesis continues even with reduced Und-P levels, cell constriction is specifically halted. We successfully exploited this knowledge to create a combination treatment strategy where the FDA-approved drug clomiphene, an inhibitor of Und-P synthesis, is paired up with amoxicillin. Our results show that clomiphene potentiates the antimicrobial activity of amoxicillin and that combination therapy resensitizes amoxicillin-resistant S. pneumoniae. These findings could provide a starting point to develop a solution for the increasing amount of hard-to-treat amoxicillin-resistant pneumococcal infections.

Streptococcus pneumoniae is a commensal bacterium of the human nasopharynx, but can cause harmful infections if it spreads to other parts of the body, such as pneumonia, sepsis or meningitis. To facilitate pathogenesis studies, we constructed a doxycycline-inducible pooled CRISPR interference (CRISPRi) library targeting all operons in protypical S. pneumoniae strain D39V. Our library design allows fitness within the pool to be assessed by a one-step PCR reaction directly followed by Illumina sequencing (CRISPRi-seq). The doxycycline-inducible CRISPRi system is tightly controllable and suitable for both bottleneck exploration and evaluation of gene fitness in vitro and in vivo. Here, we applied CRISPRi-seq to identify genetic factors important for causing pneumococcal pneumonia. Mice were infected intratracheally with our CRISPRi library and bacteria collected at 24 h (from lung) and 48 h (from both lung and blood) post-infection. CRISPRi-seq showed a critical bottleneck at 48 h after intratracheal infection, with only a few bacteria surviving the brunt of the innate immune response to cause systemic infection. However, earlier at 24 h post-infection, many significant differences in gene fitness cost between in vitro and in vivo conditions were identified, including genes encoding known and putative novel virulence factors, genes essential only in vivo, and genes essential only in vitro. A key advantage of CRISPRi-seq over traditional transposon-based genetic screens is that all genes, including essential genes, can be tested for their role in virulence and pathogenicity. The approaches developed here should be generally applicable to study infection bottlenecks and in vivo fitness for other important human and animal pathogens.

Staphylococcus aureus biofilms play important roles during infection. The main components of these biofilms are well studied; however, we lack the full understanding of factors and genes involved in regulation of biofilm formation. To screen for essential and non-essential biofilm regulatory genes in S. aureus, we have created a pooled inducible CRISPR interference library. The pooled library is designed to allow knockdown of every transcriptional unit in the S. aureus genome, thus targeting both essential and non-essential genes. We used our library in S. aureus Newman, a strain which forms structured macrocolonies on agar plates. We performed an unbiased screen of 1500 macrocolonies and found 10 macrocolonies with stably altered structures. The genotypes of these macrocolonies were determined by sequencing the single guide RNAs of the CRISPR interference system. As a proof of the validity of the approach, we identified several genes previously reported to be implicated in biofilm and macrocolony formation, including ica-genes, and metabolic genes of the TCA-cycle and gluconeogenesis. In addition, three new genes (two encoding putative enzymes and one hypothetical genes) whose depletion resulted in completely altered macrocolonies were also identified. The molecular mechanisms explaining the roles of these proteins in biofilm formation are currently under investigation.