Uncovering Endolysins against Methicillin-Resistant Staphylococcus aureus Using a Microbial Single-Cell Genome Database.

Endolysins, peptidoglycan hydrolases derived from bacteriophages (phages), are being developed as a promising alternative to conventional antibiotics. To obtain highly active endolysins, a diverse library of these endolysins is vital. We propose here microbial single-cell genome sequencing as an efficient tool to discover dozens of previously unknown endolysins, owing to its culture-independent sequencing method. As a proof of concept, we analyzed and recovered endolysin genes within prophage regions of Staphylococcus single-amplified genomes in human skin microbiome samples. We constructed a library of chimeric endolysins by shuffling domains of the natural endolysins and performed high-throughput screening against Staphylococcus aureus. One of the lead endolysins, bbst1027, exhibited desirable antimicrobial properties, such as rapid bactericidal activity, no detectable resistance development, and in vivo efficacy. We foresee that this endolysin discovery pipeline is in principle applicable to any bacterial target and boost the development of novel antimicrobial agents.

Exploring strain diversity of dominant human skin bacterial species using single-cell genome sequencing.

To understand the role of the skin commensal bacterial community in skin health and the spread of pathogens, it is crucial to identify genetic differences in the bacterial strains corresponding to human individuals. A culture-independent genomics approach is an effective tool for obtaining massive high-quality bacterial genomes. Here we present a single-cell genome sequencing to obtain comprehensive whole-genome sequences of uncultured skin bacteria from skin swabs. We recovered 281 high-quality (HQ) and 244 medium-quality single-amplified genomes (SAGs) of multiple skin bacterial species from eight individuals, including cohabiting group. Single-cell sequencing outperformed in the genome recovery from the same skin swabs, showing 10-fold non-redundant strain genomes compared to the shotgun metagenomic sequencing and binning approach. We then focused on the abundant skin bacteria and identified intra-species diversity, especially in 47 Moraxella osloensis derived HQ SAGs, characterizing the strain-level heterogeneity at mobile genetic element profiles, including plasmids and prophages. Even between the cohabiting individual hosts, they have unique skin bacterial strains in the same species, which shows microdiversity in each host. Genetic and functional differences between skin bacterial strains are predictive of in vivo competition to adapt bacterial genome to utilize the sparse nutrients available on the skin or produce molecules that inhibit the colonization of other microbes or alter their behavior. Thus, single-cell sequencing provides a large number of genomes of higher resolution and quality than conventional metagenomic analysis and helps explore the skin commensal bacteria at the strain level, linking taxonomic and functional information.

Recovery of strain-resolved genomes from human microbiome through an integration framework of single-cell genomics and metagenomics.

BACKGROUND: Obtaining high-quality (HQ) reference genomes from microbial communities is crucial for understanding the phylogeny and function of uncultured microbes in complex microbial ecosystems. Despite improvements in bioinformatic approaches to generate curated metagenome-assembled genomes (MAGs), existing metagenome binners obtain population consensus genomes but they are nowhere comparable to genomes sequenced from isolates in terms of strain level resolution. Here, we present a framework for the integration of single-cell genomics and metagenomics, referred to as single-cell (sc) metagenomics, to reconstruct strain-resolved genomes from microbial communities at once. RESULTS: Our sc-metagenomics integration framework, termed SMAGLinker, uses single-cell amplified genomes (SAGs) generated using microfluidic technology as binning guides and integrates them with metagenome-assembled genomes (MAGs) to recover improved draft genomes. We compared sc-metagenomics with the metagenomics-alone approach using conventional metagenome binners. The sc-metagenomics approach showed precise contig binning and higher recovery rates (>97%) of rRNA and plasmids than conventional metagenomics in genome reconstruction from the cell mock community. In human microbiota samples, sc-metagenomics recovered the largest number of genomes with a total of 103 gut microbial genomes (21 HQ, with 65 showing >90% completeness) and 45 skin microbial genomes (10 HQ, with 40 showing >90% completeness), respectively. Conventional metagenomics recovered one Staphylococcus hominis genome, whereas sc-metagenomics recovered two S. hominis genomes from identical skin microbiota sample. Single-cell sequencing revealed that these S. hominis genomes were derived from two distinct strains harboring specifically different plasmids. We found that all conventional S. hominis MAGs had a substantial lack or excess of genome sequences and contamination from other Staphylococcus species (S. epidermidis). CONCLUSIONS: SMAGLinker enabled us to obtain strain-resolved genomes in the mock community and human microbiota samples by assigning metagenomic sequences correctly and covering both highly conserved genes such as rRNA genes and unique extrachromosomal elements, including plasmids. SMAGLinker will provide HQ genomes that are difficult to obtain using metagenomics alone and will facilitate the understanding of microbial ecosystems by elucidating detailed metabolic pathways and horizontal gene transfer networks. SMAGLinker is available at https://github.com/kojiari/smaglinker . Video abstract.

High-Quality Draft Single-Cell Genome Sequences of Two Gammaproteobacteria Strains Sampled from Soil in a Strawberry Farm.

Here, we present high-quality draft single-cell genome sequences of Gammaproteobacteria strains BBSC-SA01 and BBSC-SA02, obtained from uncultivated cells of soil in a strawberry farm using the single-cell sequencing platform bit-MAP. These draft genomes putatively represent novel species within Gammaproteobacteria and allow further investigation into the soil microbiome.

Characterization of Sll1558 in environmental stress tolerance of Synechocystis sp. PCC 6803.

So far, the molecular mechanisms underlying the acidic-stress responses of plants are complicated and only fragmentally understood. Here, we investigated the mechanisms responsible for acidic-stress acclimation. Previously, DNA microarray analysis identified the sll1558 gene in Synechocystis sp. PCC 6803 (hereafter called Synechocystis 6803) to be upregulated following short-term acid treatment (1 h at pH 3.0). The sll1558 gene encodes uridine diphosphate-glucose pyrophosphorylase (UDP-glucose pyrophosphorylase), which catalyzes the conversion of glucose-1-phosphate into UDP-glucose. We constructed mutant cells for this gene and analyzed their phenotype. The sll1558 gene did not completely segregate in sll1558 mutant cells; thus, Sll1558 is essential for the survival of Synechocystis 6803. Besides, the partially disrupted sll1558 mutant cells were highly sensitive to acidic stress (pH 6.0) as well as other stress conditions (high salt, high osmolality, high/low temperature, and ultraviolet-B stress); the number of sll1558 transcripts increased under these conditions. UDP-glucose is used for the synthesis of various materials, such as glycolipids. From the membrane lipid composition analysis, digalactosyldiacylglycerol decreased and phosphatidylglycerol increased in the partially disrupted sll1558 mutant cells under acidic stress. These results suggest that sll1558 is important not only for the survival of Synechocystis 6803, but also for tolerance under various stress conditions.

Sll0751 and Sll1041 are involved in acid stress tolerance in Synechocystis sp. PCC 6803.

The ATP-binding cassette (ABC) transporter is a multi-subunit membrane protein complex involved in lipid transport and acid stress tolerance in the cyanobacterium Synechocystis sp. PCC 6803. This organism has two sets of three ABC transporter subunits: Slr1045 and Slr1344, Sll0751 and Sll1002, and Sll1001 and Sll1041. We previously found that Slr1045 is essential for survival under acid stress condition (Tahara et al. 2012). In the present study, we examined the participation of other ABC transporter subunits in acid stress tolerance using a deletion mutant series of Synechocystis sp. PCC 6803. Although Slr1344 is highly homologous to Slr1045, Δslr1344 cells were not susceptible to acid stress. Δsll0751 and Δsll1041 cells displayed acid stress sensitivity, whereas Δsll1001/sll1002 double mutant cells grew normally. Under high- and low-temperature stress conditions, the growth rate of Δslr1344 and Δsll1001/sll1002 cells did not differ from WT cells, whereas Δsll0751 and Δsll1041 cells showed significant growth retardation, as previously observed in Δslr1045 cells. Moreover, nile red staining showed more lipid accumulation in Δslr1045, Δsll0751, and Δsll1041 cells than in WT cells. These results suggest that Slr1045, Sll0751, and Sll1041 function together as a lipid transport complex in Synechocystis sp. PCC 6803 and are essential for growth under various stresses.

Slr2019, lipid A transporter homolog, is essential for acidic tolerance in Synechocystis sp. PCC6803.

Living organisms must defend themselves against various environmental stresses. Extracellular polysaccharide-producing cells exhibit enhanced tolerance toward adverse environmental stress. In Synechocystis sp. PCC6803 (Synechocystis), lipopolysaccharide (LPS) may play a role in this protection. To examine the relationship between stress tolerance of Synechocystis and LPS, we focused on Slr2019 because Slr2019 is homologous to MsbA in Escherichia coli, which is related to LPS synthesis. First, to obtain a defective mutant of LPS, we constructed the slr2019 insertion mutant (slr2019) strain. Sodium deoxycholate-polyacrylamide gel electrophoresis indicated that slr2019 strain did not synthesize normal LPS. Second, to clarify the participation of LPS in acid tolerance, wild type (WT) and slr2019 strain were grown under acid stress; slr2019 strain growth was significantly weaker than WT growth. Third, to examine influences on stress tolerance, slr2019 strain was grown under various stresses. Under salinity and temperature stress, slr2019 strain grew significantly slower than WT. To confirm cell morphology, cell shape and envelope of slr2019 strain were observed by transmission electron microscopy; slr2019 cells contained more electron-transparent bodies than WT cells. Finally, to confirm whether electron-transparent bodies are poly-3-hydroxybutyrate (PHB), slr2019 strain was stained with Nile Blue A, a PHB detector, and observed by fluorescence microscopy. The PHB granule content ratio of WT and slr2019 strain grown at BG-11 pH 8.0 was each 7.18 and 8.41 %. At pH 6.0, the PHB granule content ratio of WT and slr2019 strain was 2.99 and 2.60 %. However, the PHB granule content ratio of WT and slr2019 strain grown at BG-11N-reduced was 10.82 and 0.56 %. Because slr2019 strain significantly decreased PHB under BG-11N-reduced compared with WT, LPS synthesis may be related to PHB under particular conditions. These results indicated that Slr2019 is necessary for Synechocystis survival in various stresses.

Genomic analysis of parallel-evolved cyanobacterium Synechocystis sp. PCC 6803 under acid stress.

Experimental evolution is a powerful tool for clarifying phenotypic and genotypic changes responsible for adaptive evolution. In this study, we isolated acid-adapted Synechocystis sp. PCC 6803 (Synechocystis 6803) strains to identify genes involved in acid tolerance. Synechocystis 6803 is rarely found in habitants with pH < 5.75. The parent (P) strain was cultured in BG-11 at pH 6.0. We gradually lowered the pH of the medium from pH 6.0 to pH 5.5 over 3 months. Our adapted cells could grow in acid stress conditions at pH 5.5, whereas the parent cells could not. We performed whole-genome sequencing and compared the acid-adapted and P strains, thereby identifying 11 SNPs in the acid-adapted strains, including in Fo F1-ATPase. To determine whether the SNP genes responded to acid stress, we examined gene expression in the adapted strains using quantitative reverse-transcription polymerase chain reaction. sll0914, sll1496, sll0528, and sll1144 expressions increased under acid stress in the P strain, whereas sll0162, sll0163, slr0623, and slr0529 expressions decreased. There were no differences in the SNP genes expression levels between the P strain and two adapted strains, except for sll0528. These results suggest that SNPs in certain genes are involved in acid stress tolerance in Synechocystis 6803.

Sll0939 is induced by Slr0967 in the cyanobacterium Synechocystis sp. PCC6803 and is essential for growth under various stress conditions.

In this study, the genes expressed in response to low pH stress were identified in the unicellular cyanobacterium Synechocystis sp. PCC 6803 using DNA microarrays. The expression of slr0967 and sll0939 constantly increased throughout 4-h acid stress conditions. Overexpression of these two genes under the control of the trc promoter induced the cells to become tolerant to acid stress. The Δslr0967 and Δsll0939 mutant cells exhibited sensitivity to osmotic and salt stress, whereas the trc mutants of these genes exhibited tolerance to these types of stress. Microarray analysis of the Δslr0967 mutant under acid stress conditions showed that expression of the high light-inducible protein ssr2595 (HliB) and the two-component response regulator slr1214 (rre15) were out of regulation due to gene inactivation, whereas they were upregulated by acid stress in the wild-type cells. Microarray analysis and real-time quantitative reverse transcription-polymerase chain reaction analysis showed that the expression of sll0939 was significantly repressed in the slr0967 deletion mutant. These results suggest that sll0939 is directly involved in the low pH tolerance of Synechocystis sp. PCC 6803 and that slr0967 may be essential for the induction of acid stress-responsive genes.