ABSTRACT

Here, we report draft genome sequences for nine strains of “Candidatus Nanosynbacter sp. HMT-352.” These strains and their sequences were used to interrogate strain-level variations in host range, gene content, and growth dynamics among the phylum “Candidatus Saccharibacteria.”

ANNOUNCEMENT

The nine “Candidatus Nanosynbacter sp. HMT-352” (hereafter, HMT-352) strains reported here (Table 1) were recently isolated from the human oral cavity (1) and are the first members of the phylum “Candidatus Saccharibacteria,” a major lineage of the Candidate Phyla Radiation (CPR) (2), to be characterized at the strain level.
TABLE 1
TABLE 1 Summary information for the nine “Ca. Nanosynbacter sp. HMT-352” strains cultured from the human oral cavity
Strain nameBioSample accession no.SRA accession no. for:Total length (bp)No. of contigsN50 (bp)GC content (%)Completion (%)Redundancy (%)No. of genesAssembly coverage (×) for:
MonocultureCocultureMonocultureCoculture
TM7-001SAMN23492223SRR18278454SRR18278446771,8071771,80743.2583.102.828033,276432
TM7-008SAMN23492221SRR18278459SRR18278451725,5802548,42443.1784.510.007473,0632,387
TM7-053SAMN23492224SRR18278461SRR18278463755,9842546,11743.1884.514.237751,4031,097
TM7-057SAMN23492218SRR18278457SRR18278449758,4806490,40242.9984.512.828047281,156
TM7-072SAMN23492220SRR18278453SRR18278445733,2101733,21043.3184.511.417422,9111,235
TM7-075SAMN23492222SRR18278460SRR18278462730,9382448,95543.1584.514.237553,4841,767
TM7-076SAMN23492217SRR18278456SRR18278448756,0984170,97943.1983.100.007913,5191,213
TM7-087SAMN23492219SRR18278458SRR18278450741,9123536,84143.4284.510.007713,2592,736
TM7-037SAMN23492216SRR18278452SRR18278444718,2831718,28343.2683.102.82735651390
These nine HMT-352 strains were isolated from human saliva using a previously described “baiting” method (1, 3). Briefly, saliva samples were centrifuged, filtered through a 0.45-μM filter, and cocultured in brain heart infusion medium (catalog number 237500; BD, NJ, USA) with potential basibionts (bacterial hosts). The cocultures were incubated at 37°C and passaged at a dilution of 1:10 every 2 days into fresh medium. A previously described modified MasterPure DNA isolation kit (catalog number MGP04100; Epicentre, WI, USA) protocol (4) was used to isolate genomic DNA (gDNA) from both filter-isolated HMT-352 cells and the HMT-352-basibiont cocultures. Briefly, bacterial cultures were mixed with glass beads (catalog number G8772; Sigma, St. Louis, MO) and disrupted using a bead-beating homogenizer. gDNA isolation was then performed according to the manufacturer’s protocol. The gDNA was randomly fragmented by sonication and then end-polished, A-tailed, and ligated with full-length Illumina adapters. The library constructs were purified using the AMPure XP system (Beckman Coulter, IN, USA) and checked for size distribution using a 2100 Bioanalyzer (Agilent Technologies, CA, USA). The libraries were then sequenced on an Illumina NovaSeq instrument (paired-end [PE] 150-bp reads).
Default parameters were used for the computational analyses except where otherwise noted. The reads were quality controlled using iu-filter-quality-minoche from illumina-utils v2.12 (5). For each new HMT-352 strain, genomes were assembled using a previously described Anvi’o v7.1 workflow (1, 6) that employed both the isolate and coculture genomic libraries. Briefly, for each strain, libraries from the isolated HMT-352 were individually assembled using metaSPAdes v3.15.3 (7) and binned using MaxBin2 v2.2.4-1 (8). The bins were then manually refined and reassembled using both the isolate and coculture libraries. Genes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v5.3 (9). All genomes were less than 5% redundant, between 83% and 85% complete, and contained between 732 and 801 genes (Table 1).
The HMT-352 strains were compared to representative Candidatus Saccharibacteria from eHOMD v15.22 (10) using full-length 16S rRNA sequences aligned with MAFFT v7.490 (11). All strains had more than 98% homology to the closest eHOMD species. The average nucleotide identity (ANI) values over all alignable genome fractions, however, ranged between 93% and 95%, at or below the extreme end of the accepted range for intraspecies variation (1214). Such substantial intraspecies genetic diversity is additionally apparent in Fig. 1, which provides a comparison of the phylogenetic differences between the 16S rRNA, select marker gene, and single-copy core gene trees. The unexpectedly high nucleotide diversity among these strains warrants further investigation and accentuates that broad phylogenetic characterization of the CPR is the next step in understanding these bacteria.
FIG 1
FIG 1 16S rRNA, select marker gene, and single-copy core gene trees of the reported HMT-352 strains. The previously reported HMT-352 strain KC1 (15) is additionally included. The node labels represent bootstrap support. (A) Maximum-likelihood tree based on full-length 16S rRNA sequences constructed using IQ-TREE v2.1.4-beta with ultrafast bootstrap (-bb 1500) (16). Nanosynbacter lyticus strain TM7x (17) is included as an outgroup. (B) Phylogenomic tree constructed using FastTree 2 v2.1.11-1 (18) with 60 concatenated core protein amino acid sequences and TM7x (HMT-952) included as an outgroup. (C) Maximum-likelihood tree inferred using 523 concatenated single-copy core gene amino acid sequences found in all strains constructed using IQ-TREE with ultrafast bootstrap (-bb 1500).

Data availability.

Cultures of these strains are available upon request. The sequence data have been deposited at NCBI under the BioProject accession number PRJNA784561. The BioSample and SRA accession numbers are listed in Table 1. All code used to assemble and analyze the genomes is available at https://www.borlab.org/resources.

ACKNOWLEDGMENTS

We were supported by the National Institute of Dental and Craniofacial Research of the National Institutes of Health under awards 1R01DE023810, 1R01DE020102, and 1R01DE026186 (to X.H. and J.S.M.) and 5K99DE027719-02 and 5R00DE027719-04 (to B.B.). Additional support was provided to D.R.U. by Harvard University’s Department of Organismic and Evolutionary Biology program and the National Science Foundation Graduate Research Fellowship Program under grant DGE1745303.

REFERENCES

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Information & Contributors

Information

Published In

cover image Microbiology Resource Announcements
Microbiology Resource Announcements
Volume 11Number 818 August 2022
eLocator: e00403-22
Editor: Irene L. G. Newton, Indiana University, Bloomington
PubMed: 35894623

History

Received: 25 April 2022
Accepted: 11 July 2022
Published online: 27 July 2022

Contributors

Authors

Jett Liu
The Forsyth Institute, Cambridge, Massachusetts, USA
Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts, USA
Jie Nie
Department of Cariology and Endodontology, Peking University School of Stomatology, Beijing, China
Department of Periodontics, University of Washington, Seattle, Washington, USA
Department of Periodontics, University of Washington, Seattle, Washington, USA
Department of Periodontics, University of Washington, Seattle, Washington, USA
Xiaoyan Wang
Department of Cariology and Endodontology, Peking University School of Stomatology, Beijing, China
Department of Periodontics, University of Washington, Seattle, Washington, USA
The Forsyth Institute, Cambridge, Massachusetts, USA
Department of Oral Medicine, Infection, and Immunity, Harvard School of Dental Medicine, Boston, Massachusetts, USA
The Forsyth Institute, Cambridge, Massachusetts, USA
Department of Oral Medicine, Infection, and Immunity, Harvard School of Dental Medicine, Boston, Massachusetts, USA

Editor

Irene L. G. Newton
Editor
Indiana University, Bloomington

Notes

Jie Nie and Daniel R. Utter contributed equally to this article. Author order was determined alphabetically by first name.
The authors declare no conflict of interest.

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