Background: Lanthipeptides belong to the ribosomally synthesized and post-translationally modified peptide group of natural products and have a variety of biological activities ranging from antibiotics to antinociceptives. These peptides are cyclized through thioether crosslinks and can bear other secondary post-translational modifications. While lanthipeptide biosynthetic gene clusters can be identified by the presence of genes encoding characteristic enzymes involved in the post-translational modification process, locating the precursor peptides encoded within these clusters is challenging due to their short length and high sequence variability, which limits the high-throughput exploration of lanthipeptide biosynthesis. To address this challenge, we enhanced the predictive capabilities of Rapid ORF Description & Evaluation Online (RODEO) to identify members of all four known classes of lanthipeptides. Results: Using RODEO, we mined over 100,000 bacterial and archaeal genomes in the RefSeq database. We identified nearly 8500 lanthipeptide precursor peptides. These precursor peptides were identified in a broad range of bacterial phyla as well as the Euryarchaeota phylum of archaea. Bacteroidetes were found to encode a large number of these biosynthetic gene clusters, despite making up a relatively small portion of the genomes in this dataset. A number of these precursor peptides are similar to those of previously characterized lanthipeptides, but even more were not, including potential antibiotics. One such new antimicrobial lanthipeptide was purified and characterized. Additionally, examination of the biosynthetic gene clusters revealed that enzymes installing secondary post-translational modifications are more widespread than initially thought. Conclusion: Lanthipeptide biosynthetic gene clusters are more widely distributed and the precursor peptides encoded within these clusters are more diverse than previously appreciated, demonstrating that the lanthipeptide sequence-function space remains largely underexplored.
Multiple epidemiological studies identify Dolosigranulum pigrum as a candidate beneficial bacterium based on its positive association with health, including negative associations with nasal/nasopharyngeal colonization by the pathogenic species Staphylococcus aureus and Streptococcus pneumoniae. Using a multipronged approach to gain new insights into D. pigrum function, we observed phenotypic interactions and predictions of genomic capacity that support the idea of a role for microbe-microbe interactions involving D. pigrum in shaping the composition of human nasal microbiota. We identified in vivo community-level and in vitro phenotypic cooperation by specific nasal Corynebacterium species. Also, D. pigrum inhibited S. aureus growth in vitro, whereas robust inhibition of S. pneumoniae required both D. pigrum and a nasal Corynebacterium together. D. pigrum l-lactic acid production was insufficient to account for these inhibitions. Genomic analysis of 11 strains revealed that D. pigrum has a small genome (average 1.86 Mb) and multiple predicted auxotrophies consistent with D. pigrum relying on its human host and on cocolonizing bacteria for key nutrients. Further, the accessory genome of D. pigrum harbored a diverse repertoire of biosynthetic gene clusters, some of which may have a role in microbe-microbe interactions. These new insights into D. pigrum’s functions advance the field from compositional analysis to genomic and phenotypic experimentation on a potentially beneficial bacterial resident of the human upper respiratory tract and lay the foundation for future animal and clinical experiments. IMPORTANCE Staphylococcus aureus and Streptococcus pneumoniae infections cause significant morbidity and mortality in humans. For both, nasal colonization is a risk factor for infection. Studies of nasal microbiota identify Dolosigranulum pigrum as a benign bacterium present when adults are free of S. aureus or when children are free of S. pneumoniae. Here, we validated these in vivo associations with functional assays. We found that D. pigrum inhibited S. aureus in vitro and, together with a specific nasal Corynebacterium species, also inhibited S. pneumoniae. Furthermore, genomic analysis of D. pigrum indicated that it must obtain key nutrients from other nasal bacteria or from humans. These phenotypic interactions support the idea of a role for microbe-microbe interactions in shaping the composition of human nasal microbiota and implicate D. pigrum as a mutualist of humans. These findings support the feasibility of future development of microbe-targeted interventions to reshape nasal microbiota composition to exclude S. aureus and/or S. pneumoniae.
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a promising source of new antimicrobials in the face of rising antibiotic resistance. Here, we report a scalable platform that combines high-throughput bioinformatics with automated biosynthetic gene cluster refactoring for rapid evaluation of uncharacterized gene clusters. As a proof of concept, 96 RiPP gene clusters that originate from diverse bacterial phyla involving 383 biosynthetic genes are refactored in a high-throughput manner using a biological foundry with a success rate of 86%. Heterologous expression of all successfully refactored gene clusters in Escherichia coli enables the discovery of 30 compounds covering six RiPP classes: lanthipeptides, lasso peptides, graspetides, glycocins, linear azol(in)e-containing peptides, and thioamitides. A subset of the discovered lanthipeptides exhibit antibiotic activity, with one class II lanthipeptide showing low µM activity against Klebsiella pneumoniae, an ESKAPE pathogen. Overall, this work provides a robust platform for rapidly discovering RiPPs.
25Background: Multiple epidemiological studies identify Dolosigranulum pigrum as a 26 candidate beneficial bacterium based on its positive association with health, including 27 negative associations with nasal/nasopharyngeal colonization by the pathogenic 28 species Staphylococcus aureus and Streptococcus pneumoniae. 29Results: Using a multipronged approach to gain new insights into D. pigrum function, 30 we observed phenotypic interactions and predictions of genomic capacity that support a 31 role for microbe-microbe interactions involving D. pigrum in shaping the composition of 32 human nasal microbiota. We identified in vivo community-level and in vitro phenotypic 33 cooperation by specific nasal Corynebacterium species. Also, D. pigrum inhibited S. 34 aureus growth in vitro. Whereas, robust inhibition of S. pneumoniae required both D. 35 pigrum and a nasal Corynebacterium together, and not either alone. D. pigrum L-lactic- 36 acid production was insufficient to account for these inhibitions. Genomic analysis of 11 37 strains revealed that D. pigrum has a small genome (average 1.86 Mb) and multiple 38 predicted auxotrophies consistent with D. pigrum relying on its human host and 39 cocolonizing bacteria for key nutrients. Further, the accessory genome of D. pigrum 40 3 encoded a diverse repertoire of biosynthetic gene clusters, some of which may have a 41 role in microbe-microbe interactions. 42 Conclusions: These new insights into D. pigrum's functions advance the field from 43 compositional analysis to genomic and phenotypic experimentation on a potentially 44 beneficial bacterial resident of the human upper respiratory tract and lay the foundation 45 for future animal and clinical experiments. 46 47 48 Streptococcus pneumoniae, microbe-microbe interactions, interspecies interactions, 50 upper respiratory tract, nasal, microbiota, comparative genomics 51 4 Background 52 Colonization of the human nasal passages by Staphylococcus aureus or Streptococcus 53pneumoniae is a major risk factor for infection by the colonizing bacterium at a distant 54 body site [1][2][3][4][5]. Interventions that reduce the prevalence of colonization also reduce the 55 risk of infection and transmission, e.g., as in [6, 7]. S. aureus and S. pneumoniae are 56 major human pathogens that cause significant morbidity and mortality worldwide [8][9][10][11]. 57 There are also concerns regarding rising rates of antimicrobial resistance [12] and the 58 potential for long-term effects of antibiotics early in life [13]. Thus, efforts have recently 59 focused on the identification of candidate bacteria that confer colonization resistance 60 against S. aureus [14-21] and S. pneumoniae [22-25], with particular urgency for S. 61 aureus in the absence of an effective vaccine. 62 Dolosigranulum pigrum has emerged in multiple studies of the human upper respiratory 63 tract microbiota, colonizing with or without Corynebacterium species, as potentially 64 beneficial and/or protective against colonization by S. aureus and ...
Dolosigranulum pigrum is a candidate beneficial bacterium with potential for future therapeutic use. This is based on its positive associations with characteristics of health in multiple studies of human nasal microbiota across the span of human life.
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