DOI: 10.5061/dryad.g4f4qrfx7
title: Supplementary materials for Phylogenomics and genetic analysis of solvent-producing Clostridium species

description: The genus Clostridium is a large and diverse group within the Bacillota
 (formerly Firmicutes), whose members can encode useful complex traits such
 as solvent production, gas-fermentation, and lignocellulose breakdown. We
 describe 270 genome sequences of solventogenic clostridia from a
 comprehensive industrial strain collection assembled by Professor David
 Jones that includes 194 C. beijerenckiI, 57 C. saccharobutylicum,
 4 C. saccharoperbutylacetonicum, 5 C. butyricum, 7
 C. acetobutylicum, and 3 C. tetanomorphum genomes. We report
 methods, analyses and characterization for phylogeny, key attributes, core
 biosynthetic genes, secondary metabolites, plasmids, prophage/CRISPR
 diversity, cellulosomes and quorum sensing for the 6 species. The expanded
 genomic data described here will facilitate engineering of
 solvent-producing clostridia as well as non-model microorganisms with
 innately desirable traits. Sequences could be applied in conventional
 platform biocatalysts such as yeast or Escherichia coli for enhanced
 chemical production. Recently, gene sequences from this collection were
 used to engineer Clostridium autoethanogenum, a gas-fermenting autotrophic
 acetogen, for continuous acetone or isopropanol production, as well as
 butanol, butanoic acid, hexanol and hexanoic acid production. 
description: A modified phenol-chloroform extraction based on Annan et al.
 (2019) was used to purify genomic DNA. Cells were harvested by
 centrifugation at 6,000 <em>x</em> g for 15 min at 4°C, and
 washed in 50 mL potassium phosphate buffer (10 mM, pH 7.5). The cells were
 resuspended in STE buffer (50 mM Tris-HCl, pH 8.0; 1 mM EDTA; 200 mM
 sucrose) with 30 μL lysozyme (100 mg/mL), followed by a combination of
 SDS, EDTA, Tris, RNaseA/T1, and Proteinase K. Each addition was followed
 by specific incubation steps at 37°C. The lysate was processed using Phase
 Lock Gel Heavy tubes and a modified phenol::chloroform procedure that
 employed MaXtract High Density (Qiagen) phase-lock tubes. DNA was
 precipitated, then washed with -20°C 70% ethanol, dried, and resuspended
 in heated (60°C) 1x TE buffer (10 mM Tris, pH 8, and 0.1 mM EDTA).
 Purified DNA underwent 16S rRNA gene sequencing to confirm strain identity
 before being shipped to the DOE Joint Genome Institute (JGI) for genome
 sequencing and annotation. <strong>Reference:</strong> Annan
 FJ, et al. Appl Microbiol Biotechnol. 2019 Jun;103(11):4633-4648. Genomes
 were sequenced at the JGI using Pacific Biosciences (PacBio) technology on
 either an RSII instrument (P6/C4 chemistry), or Sequel (v2.1 v2 or v3.0
 chemistries) or using an Illumina NovaSeq (v1 chemistry) and have been
 reported previously (https://pubmed.ncbi.nlm.nih.gov/37764097/). All genome sequences were annotated using the JGI IMG pipeline, with the vast majority by version v.5.0.10 and details available for each at the JGI IMG database. Genomes used in this study are curated<strong> </strong>under the GOLD study ID Gs0118866, with links to raw sequence data available via JGI or via the NCBI SRA database. Contigs were classified into plasmid and chromosomal categories to determine plasmid presence or absence using PlasFlow v1.1.0. For convenience, the 270 sub-projects are deposited under NCBI BioProject PRJNA990349. Strains analyzed in this study are shown Supplementary Table 1, and include relevant culture collection details (Supplementary Tables 2-3). Additional methodological details are provided in accepted Scientific Data manucript SDATA-23-01651.
description: # Supplementary Dataset and Tables for Phylogenomics and Genetic Analysis
 of Solvent-Producing *Clostridium* Species This Dryad submission supports
 a publication entitled “Phylogenomics and Genetic Analysis of
 Solvent-Producing *Clostridium* Species” accepted for publication at
 *Scientific Data* (SDATA-23-01651) in April 2024
 ([https://www.nature.com/sdata/)](https://www.nature.com/sdata/\)). The
 paper describes 270 genome sequences and detailed analyses. The raw
 sequence and genome sequence information are publicly available, described
 in the publication and in the related works section of this submission.
 This Dryad dataset provides supplementary information to the *Scientific
 Data* publication. ## Description of the data and file structure There are
 11 files in this dataset in excel format: **Supplementary Table 1**. This
 file contains 270 DJ strain designations, linked to database references
 (e.g. GenBank accession numbers, Genome IDs) and provides important genome
 analysis results such as CheckM2 outputs, contig and gene numbers. Header
 information is provided in row 1 and information is organized by strain
 designation in ascending order. **Supplementary Table 2**. A subset of
 strains whose genomes are described in the *Sci Data* paper are available
 from international culture collection are listed in this table (colored
 red). The table is arranged by phylogenetic clade and by species/strain
 designations and it also provides other strains for multiple species that
 are also deposited in international culture collections (colored black).
 The table contents for different species contains several blank cells that
 separate species for visual ease of access. **Supplementary Table 3**. The
 National Chemical Products (NCP) chemical and fermentation company
 operated industrial ABE fermentation processes in South Africa from 1935
 to 1983. This table provides cross reference information for newly
 generated genome sequence information to historical NCP designations.
 Header information is provided in row 1. Information is organized by DJ
 strain designation in ascending order, followed NCP information, then full
 strain name and associated JGI IMG Genome ID separated by a comma.
 **Supplementary Table 4**. Core to the clostridial acetone-butanol-ethanol
 (ABE) process are key biosynthesis proteins, which are listed in this
 table. Type strain protein sequences used in the Sci Data paper as input
 search sequences are listed, along with search output results organized by
 individual enzymes, protein sequence and by strain hit results. The table
 contains several blank cells that separate enzymes for visual ease of
 access. **Supplementary Table 5**. BlastP analysis of RRNPP-type
 quorum-sensing systems. Table S5a: Blast hits corresponding to in putative
 RRNPP-type quorum-sensing system in seven *C. acetobutylicum* genomes.
 Below the first table, separated by a blank row is Table S5b: Blast hits
 corresponding to in putative RRNPP-type quorum-sensing system in seven *C.
 saccharoperbutylacetonicum* genomes. The genome columns indicate the type
 strain reference input sequence used to search DJ genomes, which are
 described in the references provided in the table. Putative hits to DJ
 strains are shown per genome and protein accession numbers are provided.
 **Supplementary Table 6**. Summary of cellulosomal genes in
 butanol-producing clostridia. The presence of sequences for cohesin(s) or
 dockerin in the given gene is indicated by Coh(s) or Doc, respectively.
 The genes are organized in a cluster, except for the underlined genes that
 are scattered through the respective genomes. From all the genomes
 analyzed, cellulosomal elements were retrieved only in *C. acetobutylicum,
 C. saccharoperbutylacetonicum* and one of the *C. beijerinckii* strains.
 Analysis of the seven *C. acetobutylicum* strains revealed that each
 genome contains 6 cohesins (5 in the scaffoldin sequence and one in the
 orfX gene) and 10 dockerins, as described previously for type strain ATCC
 824 (Lopez-Contreras, A.M. et al, 2003). Dockerin-containing proteins
 include the following glycoside hydrolase (GH) catalytic modules; three
 GH5s, four GH9s, one GH44, one GH48 and one GH74. Nine of these twelve
 cellulosomal genes are organized in a gene cluster, identical to that
 reported previously for *C. acetobutylicum* strains (Lopez-Contreras, A.M.
 et al, 2003; Dassa, B. et al, 2017). Similarly, the analysis of the four
 *C. saccharoperbutylacetonicum* genomes revealed a similar cellulosomal
 organization as in strain N1-4 (Levi Hevroni, B., et al, 2020), including
 2 cohesins and 8 dockerins. The annotation of the dockerin-containing
 enzymes is also analogous to those of the N1-4 strain, with two GH5s, two
 GH9s, one GH26, one GH44, one GH48 and one GH74. Moreover, five of the
 genes encoding the putative enzymes are organized in a gene cluster,
 together with the two-cohesin (bivalent) scaffoldin gene. *C.
 beijerinckii* DJ015 appeared to contain two cellulosomal genes, highly
 similar to the *C. saccharoperbutylacetonicum* genes encoding ScaA and
 GH48 (99 and 100% sequence identities, respectively). However, as a PCR
 assay was unable to confirm the presence of the genes for ScaA and GH48 in
 strain DJ015. Strain DJ015 was generated from paired-end Illumina data,
 contained 665 annotated contigs, a greater number of total contigs and was
 excluded from this study, as were several other genomes. **Supplementary
 Table 7**. Distribution of glycoside hydrolases (GHs) identified in DJ
 strains. Annotation of glycoside hydrolases was performed with dbcan2 (Han
 Zhang, Tanner Yohe, Le Huang, Sarah Entwistle, Peizhi Wu, Zhenglu Yang,
 Peter K Busk, Ying Xu, Yanbin Yin, dbCAN2: a meta server for automated
 carbohydrate-active enzyme annotation, *Nucleic Acids Research*, Volume
 46, Issue W1, 2 July 2018, Pages W95–W101)
 [https://doi.org/10.1093/nar/gky418](https://doi.org/10.1093/nar/gky418).
 The number of different GH families of enzymes (described by dbcan2) is
 shown per genome (headers). Blank cells indicate no hit identified.
 **Supplementary Table 8**. Putative *in silico* plasmid detection in DJ
 genomes. Contigs were classified into plasmid and chromosomal categories
 to determine plasmid presence or absence using PlasFlow v1.1.0. Header
 information is provided in row 1. Information is organized by DJ strain,
 putative detection (Yes or No) in the PlasFlow Plasmid Call header,
 followed by predicted plasmid number of different plasmids per genome
 (Predicted Plasmid Count), followed by plasmid size estimates. Where
 plasmids were not identified a "-" value is present.
 **Supplementary Table 9**. Analysis of the DJ genomes identified a number
 of prophage sequences, which are listed. The table includes taxonomic
 affiliation (prophage, host species, genomic boundaries, NCBI RefSeq &
 Marker genes) using the IMG/VR approach as well as vContact clusters,
 along with quality information obtained with CheckV. The host genome,
 contig number and genomic boundaries are described under the Prohage
 header, respectively, separated by bars. Headers are provided in row one
 for other attributes. The table contents several blank cells where there
 was no hit or information or for visual ease of access. **Supplementary
 Table 10**. CRISPR-Cas systems for ABE clostridia genomes were analyzed
 for prevalence, diversity, and dynamics in closely related host species.
 The complete Type I-B system was the most common, detected in all the *C.
 saccharobutylicum*, *C. saccharoperbutylacetonicum*, and *C.
 tetanomorphum* genomes, and a minority of genomes from *C. butyricum* and
 *C. beijerinckii*. This dataset identifies CRISPR-Cas systems, arranged by
 hits to host genome species, and contains details such as the number of
 spacer sequences, locations, repeat sequences as named in row 1.
 **Supplementary Table 11**. List of matches between industrial clostridia
 CRISPR spacers and phage sequences. Information about phage sequences was
 obtained either from this study or from the IMG/VR v3 database. Row 1 of
 this dataset has descriptive column headers that identify attributes of
 sequence matches. The host genome (JGI IMG Genome ID), contig number and
 starting genome coordinate are described under the Spacer header,
 respectively, separated by bars. Host species are identified, followed
 array name and additional characteristics such as phage match, ecosystem.
 ## Sharing/Access information The source for the underlying genome and
 sequence data is described in: Microorganisms. 2023 Sep 7;11(9):2253. doi:
 10.3390/microorganisms11092253. Solvent-Producing Clostridia Revisited.
 David T Jones , Frederik Schulz , Simon Roux , Steven D Brown.
 [https://pubmed.ncbi.nlm.nih.gov/37764097/](https://pubmed.ncbi.nlm.nih.gov/37764097/) For convenience, the 270 sub-projects are deposited under NCBI BioProject PRJNA990349.