A computational protocol to characterize elusive Candidate Phyla Radiation bacteria in oral environments using metagenomic data

Abstract

24Several studies have documented the diversity and potential pathogenic associations of 25 organisms in the human oral cavity. Although much progress has been made in 26 understanding the complex bacterial community inhabiting the human oral cavity, our 27 understanding of some microorganisms is less resolved due to a variety of reasons. One 28 such little-understood group is the candidate phyla radiation (CPR), which is a recently 29 identified, but highly abundant group of ultrasmall bacteria with reduced genomes and 30 unusual ribosomes. Here, we present a computational protocol for the detection of CPR 31 organisms from metagenomic data. Our approach relies on a self-constructed dataset 32 comprising published CPR genomic sequences as a filter to identify CPR sequences from 33 metagenomic sequencing data. After assembly and functional prediction, the taxonomic 34 affiliation of CPR contigs can be identified through phylogenetic analysis with publically 35 available 16S rRNA gene and ribosomal proteins, in addition to sequence similarity 36 analyses (e.g., average nucleotide identity calculations and contig mapping). Using this 37 protocol, we reconstructed two draft genomes of organisms within the TM7 superphylum, 38 that had genome sizes of 0.594 Mb and 0.678 Mb. Among the predicted functional genes 39 of the constructed genomes, a high percentage were related to signal transduction, cell 40 motility, and cell envelope biogenesis, which could contribute to cellular morphological 41 changes in response to environmental cues.42 3 Importance 43 Candidate phyla radiation (CPR) bacterial group is a recently identified, but highly 44 diverse and abundant group of ultrasmall bacteria exhibiting reduced genomes and 45 limited metabolic capacities. A number of studies have reported their potential pathogenic 46 associations in multiple mucosal diseases including periodontitis, halitosis, and 47 inflammatory bowel disease. However, CPR organisms are difficult to cultivate and are 48 difficult to detect with PCR-based methods due to divergent genetic sequences. Thus, our 49 understanding of CPR has lagged behind that of other bacterial component. Here, we 50 used metagenomic approaches to overcome these previous barriers to CPR identification, 51 and established a computational protocol for detection of CPR organisms from 52 metagenomic samples. The protocol describe herein holds great promise for better 53 understanding the potential biological functioning of CPR. Moreover, the pipeline could 54 be applied to other organisms that are difficult to cultivate. 55 56 Keywords: candidate phyla radiation (CPR), metagenomics, bioinformatics, 57 computational protocol 58 59 60 61 62 63 4 Introduction 64The human oral cavity is one of the five primary microbial microecosystems within 65 humans, and has been used as a model system for microbiome research (1). Dysbiosis of 66 the oral microbial community has been observed in relation to some common systemic 67 diseases including rheumatoid arthritis (2) and type 2 diab...

Microbial Symphony: Navigating the Intricacies of the Human Oral Microbiome and Its Impact on Health

“…This hitherto undiscovered CPR group of organisms showcases a remarkable diversity, encompassing over 35 distinct phyla, inhabiting a wide range of ecological niches. They also lack the genes responsible for encoding a CRISPR/Cas bacteriophage defense system [ 45 , 46 ].…”

“…This is achieved through their ability to control the structural arrangement and operational behavior of the oral microbiome. These factors have been shown to have correlations with oral diseases, such as periodontitis and halitosis [ 45 , 46 , 57 ].…”

“…The prevailing bacterial phylogenetics of the human oral microbiota, as derived from the Human Oral Microbiome Database (HOMD), can be summarized as follows [39][40][41][42][43]: The Candidate Phyla Radiation (CPR) is a large group of bacterial lineages that lack pure isolate cultures and are primarily defined through genome-resolved metagenomics [44]. CPR organisms exhibit common characteristics such as possessing small genomes and physical sizes, containing archaeal-specific RuBisCO genes, lacking certain metabolic enzymes, containing self-splicing introns within the 16S rRNA gene, and occupying deep subsidiaries within the bacterial subtree of life [45].…”

Using Genomics to Decipher the Enigmatic Properties and Survival Adaptation of Candidate Phyla Radiation

“…Two years after a CPR sequence was first detected, in 1998, the 16S rRNA phylogenetic tree revealed CPR sequences as a distinct phylum independent of archaea and eukaryotes but related to bacteria ( Figure 1 ) [ 31 ]. Due to significant divergent genetic sequences, some studies have shown the inadequacy of using 16S rRNA for CPR classification [ 7 , 32 ]. However, the use of other conserved coding genes, such as ribonucleotide reductase (RNR), topoisomerase IIA (Topo IIA), elongation factor 1 (EF1), DNA-dependent RNA polymerase subunit II (RNAP II), thymidylate synthase (ThyA), and DNA polymerase, has ultimately resulted in a very similar classification of CPR genomes [ 32 , 33 ].…”

“…Due to significant divergent genetic sequences, some studies have shown the inadequacy of using 16S rRNA for CPR classification [ 7 , 32 ]. However, the use of other conserved coding genes, such as ribonucleotide reductase (RNR), topoisomerase IIA (Topo IIA), elongation factor 1 (EF1), DNA-dependent RNA polymerase subunit II (RNAP II), thymidylate synthase (ThyA), and DNA polymerase, has ultimately resulted in a very similar classification of CPR genomes [ 32 , 33 ]. Thus, a recent genomic analysis conducted by Megrian et al showed the conserved presence of division and cell wall (dcw) genes with the necessary order (synteny) in over 1000 bacterial genomes, including CPRs [ 34 ].…”