The novel bacterial phylum Calditrichaeota is diverse, widespread and abundant in

marine sediments and has the capacity to degrade detrital proteins

This is the accepted version of the following article: Marshall IPG, Starnawski P, Cupit C,

Cáceres EF, Ettema TJG, Schramm A, et al. (2017). The novel bacterial phylum Calditrichaeota

is diverse, widespread and abundant in marine sediments and has the capacity to degrade

detrital proteins. Environmental Microbiology Reports. e-pub ahead of print, doi:

10.1111/1758-2229.12544., which has been published in final form at

http://onlinelibrary.wiley.com/doi/10.1111/1758-2229.12544/abstract. This article may be

used for non-commercial purposes in accordance with the Wiley Self-Archiving Policy

[ http://olabout.wiley.com/WileyCDA/Section/id-828039.html ].

Running head: Expansion of Calditrichaeota diversity

Ian P. G. Marshall1*, Piotr Starnawski1§*, Carina Cupit1, Eva Fernández Cáceres2, Thijs J. G.

Ettema2, Andreas Schramm1, Kasper U. Kjeldsen1

1. Center for Geomicrobiology, Section for Microbiology, Department of Bioscience,

Aarhus University, Denmark

2. Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala

University, Sweden

§Current address. Department of Molecular Medicine, Aarhus University Hospital, Denmark

* These authors contributed equally to this manuscript

Corresponding author:

Ian P.G. Marshall

Center for Geomicrobiology

Section for Microbiology, Department of Bioscience, Aarhus University

Ny Munkegade 114

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8000 Aarhus C

Denmark

E-mail: ianpgm@bios.au.dk

Telephone: +45 8715 6553

Fax: +45 8715 4326

Summary

Calditrichaeota is a recently recognised bacterial phylum with three cultured representatives,

isolated from hydrothermal vents. Here we expand the phylogeny and ecology of this novel

phylum with metagenome-derived and single-cell genomes from six uncultivated bacteria

previously not recognised as members of Calditrichaeota. Using 16S rRNA gene sequences

from these genomes, we then identified 322 16S rRNA gene sequences from cultivation-

independent studies that can now be classified as Calditrichaeota for the first time. This

dataset was used to re-analyse a collection of 16S rRNA gene amplicon datasets from marine

sediments showing that the Calditrichaeota are globally distributed in the seabed at high

abundance, making up to 6.7% of the total bacterial community. This wide distribution and

high abundance of Calditrichaeota in cold marine sediment has gone unrecognised until now.

All Calditrichaeota genomes show indications of a chemoorganoheterotrophic metabolism

with the potential to degrade detrital proteins through the use of extracellular peptidases.

Most of the genomes contain genes encoding proteins that confer O2 tolerance, consistent with

the relatively high abundance of Calditrichaeota in surficial bioturbated part of the seabed

and, together with the genes encoding extracellular peptidases, suggestive of a general

ecophysiological niche for this newly recognised phylum in marine sediment.

Introduction

The bacterial phylum Calditrichaeota has been recently recognised as an independent

phylum-level clade (Kublanov et al., 2017) with three cultured representatives in the genera

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Caldithrix (Miroshnichenko et al., 2003; 2010) and Calorithrix (Kompantseva et al., 2016).

These bacteria are anaerobic organoheterotrophic thermophiles isolated from marine

hydrothermal vents with a shared ability to degrade proteinaceous substrates. The 14-year

lag in between the phylum’s first isolate and a formal description of the phylum has meant

that Calditrichaeota has gone unrecognised in in the taxonomic classification of DNA

sequences from uncultivated microbes submitted to online databases, including

Genbank/ENA/DDBJ and the Silva small subunit ribosomal RNA (SSU rRNA) database (Quast

et al., 2013). This means that Calditrichaeota’s global distribution and abundance are

unknown and no unified view of the phylum’s unifying physiological characteristics exists

Here we used phylogenomic methods to clearly delineate the phylum Calditrichaeota and to

re-examine the taxonomic placement of several uncultured bacteria that were related to but

not previously identified as Calditrichaeota, based on genomes derived from single-cell

sequencing and metagenomic binning. We then used the 16S rRNA genes from isolate- and

uncultivated-source genomes to identify sequences in the Silva SSU rRNA database that ought

to be classified as Calditrichaeota. Since many of these were derived from marine sediments

we furthermore analysed NGS amplicon datasets from 18 different marine sediment sites to

determine the prevalence of Calditrichaeota in marine sediment. Finally, we aimed to

determine how widely the physiological traits of the three Calditrichaeota isolates are shared

with their uncultivated relatives, and identify the functions that typify this novel phylum.

Results and Discussion

Calditrichaeota was recently recognised as a bacterial phylum within the Fibrobacteres-

Chlorobi-Bacteroidetes (FCB) superphylum, with the closest relative phylum being

Ignavibacteria (Kublanov et al., 2017). Following other researchers’ approaches for defining

phyla based on genomic data (Yarza et al., 2014; Anantharaman et al., 2016), we set out to

determine whether several relatives of the genera Caldithrix and Calorithrix belonged to the

Calditrichaeota based on two criteria: (1) 16S ribosomal RNA sequence identity between

phylum members should be 75% or greater, and (2) the phylum should constitute a

monophyletic clade. Our analyses reveal that both of these criteria are fulfilled, supporting the

hypothesis that the Caldithrix and Calorithrix, alongside the genomes of two uncultivated

bacteria derived from metagenomes (RBG_16_48_16 from a terrestrial aquifer

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(Anantharaman et al., 2016) and SM23_31 from marine sediment (Baker et al., 2015)), belong

to an independent phylum based on 16S rRNA gene phylogeny, albeit with very poor

bootstrap support (48% bootstrap support, Figure 1A – see supplemental materials and

methods for a complete description). We therefore constructed a phylogenetic tree based on a

concatenated alignment of 17 single-copy orthologous protein sequences from 55 bacterial

genomes (Figure 1B – see supplemental methods for the full criteria and methods used to

choose these protein sequences). This protein-sequence-derived phylogenetic tree places

Caldithrix and six related metagenome- and single-cell-derived genomes (Table 1) in a clade

with 100% bootstrap support. Attempting to form a phylum-level clade from a more deeply

branching point in the tree would include the phylum Bacteroidetes, which contains

representatives with less than 75% 16S rRNA gene sequence identity to Caldithrix abyssi. This

means that, according to our defined criteria, six uncultured bacterial species belong to the

phylum Calditrichaeota and have not been recognised as such prior to this study.

The previous lack of recognition for the Calditrichaeota means that there are large numbers of

16S ribosomal RNA sequences obtained through cultivation-independent sequencing that

have been misassigned to phyla other than Calditrichaeota. We identified some of these by

taking all 16S rRNA gene sequences from the Silva SSU REF database v. 128 (Quast et al.,

2013) that were greater than 80% identical to the Caldithrix abyssi sequence (71051

sequences total) and constructing a FastTree2 maximum likelihood phylogenetic tree from

these sequences. From this tree we could define a set of 322 sequences that belong within the

phylum Calditrichaeota (Supplemental Figure SF1A, Supplemental Table ST1). Silva taxonomy

placed almost all of these sequences within the phyla Fibrobacteres, Deferribacteres, and LCP-

89, while Greengenes taxonomy mostly showed provisional recognition of the “Caldithrix”

phylum and a number of unclassified sequences (Supplemental Figure SF1B, Supplemental

Table ST1). In contrast to the source of Caldithrix and Calorithrix isolates, the majority of these

sequences were derived from non-hydrothermal marine sediment. We therefore identified

reads from 18 different seabed sites accessible through publicly available next-generation 16S

rRNA gene amplicon community analysis studies of diffusive marine sediment environments

that were 95% identical or greater to one of the 322 representative Calditrichaeota

sequences. This analysis showed that Calditrichaeota is globally distributed and highly

abundant in marine sediment (Supplemental Table ST2, Figure 2A), comprising up to 6.7% of

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16S rRNA gene amplicon libraries. The sample with the highest fraction of Calditrichaeota was

a sediment sample from a depth of 0-4cm in the Caspian Sea (Mahmoudi et al., 2015).

Calditrichaeota appear to be more abundant in shallow sediments (<1 meters below seafloor

[mbsf]) than in the deep subsurface, even though Calditrichaeota sequences were found down

to 141 mbsf (Figure 2B, Supplemental Table ST2).

We next examined what physiological features might be common across the phylum (see

Supplemental Table ST3 for an overview of inferred physiological characteristics). The three

cultured representatives of the Calditrichaeota, Caldithrix abyssi, Caldithrix palaeochoryensis,

and Calorithrix insularis, share certain features: they are all anaerobic thermophiles capable of

chemoorganoheterotrophic growth, in particular the fermentation of proteinaceous

compounds and the production of H2. All of the seven Calditrichaeota genomes have genes

encoding extracellular peptidases (Figure 3, Supplemental Table ST4), with such genes

constituting 8-75% (median 29%) of their complement of genes that encode proteins with a

predicted extracellular localization. The most common extracellular peptidase family is the

S8A (subtilisin) family, involved in the extracellular breakdown of proteins for use as a

substrate by the cell (Rawlings et al., 2014). All genomes also contain genes encoding proteins

for peptide or amino acid uptake (Figure 3, Supplemental Table ST6). Six of the seven

Calditrichaeota genomes have genes encoding NiFe hydrogenases, suggesting a capacity for

either H2 production during fermentation or the use of H2 as an electron donor. The presence

of genes encoding extracellular peptidases and amino acid/oligopeptide transport in all seven

Calditrichaeota genomes, and the presence of hydrogenases in six of the genomes, suggest

that the ability to ferment proteinaceous substrates and produce H2 is a broad defining

feature of the phylum Calditrichaeota beyond the two genera isolated to date.

Genes encoding cytochrome c oxidase were present in four of the genomes, including that of

the strictly anaerobic C. abyssi. This suggests that these enzymes could be involved in either

protection from oxidation by O2, as has been observed in other strict anaerobes (Lamrabet et

al., 2011; Ramel et al., 2013; 2015), or in aerobic metabolism. Genes encoding catalase (2/7

genomes), peroxidase (4/7 genomes), and superoxide dismutase (4/7 genomes) were also

widespread, with all but one genome (AABM5.125.24) encoding some mechanism for O2

protection (Supplemental Table ST3). An O2-tolerant or aerobic lifestyle is consistent with the

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highest abundances of Calditrichaeota found in sediment at depths shallower than 1 mbsf

(Figure 2B), as O2 can penetrate surficial sediment by bioturbation (Kristensen et al., 2012).

Interestingly, the only genome in our analysis without any genes encoding O2-protection

enzymes or cytochrome C oxidase was from the deepest marine sediment depth that

environmental genomes were obtained from (1.25 mbsf, Table 1) and therefore the least

likely to be exposed to O2.

There are also certain characteristics that differentiate the three cultivated Calditrichaeota,

and the other genomes in this phylum reflect this metabolic diversity. Only C. abyssi and C.

insularis are capable of respiratory growth with nitrate as electron acceptor and

chemolithoheterotrophic growth on H2, while only C. palaeochoryensis can ferment

polysaccharides (and produce a wider range of fermentation products) (Miroshnichenko et

al., 2010; Kompantseva et al., 2016). A gene encoding nitrate reductase was only identified in

C. abyssi, suggesting that nitrate reduction is not necessarily a widespread trait within

Calditrichaeota, although a nitrite reductase was also identified in Caldithrix sp. RBG 13_44_9

(Supplemental Table ST3). Extracellular glycoside hydrolases were identified in four of the

seven genomes (Supplemental Table ST5).

This study has revealed the heretofore hidden extent of the novel phylum Calditrichaeota in

marine sediment by critical re-analysis of previously sequenced genomes and 16S rRNA

sequences from uncultivated bacteria. A re-classification of these databases is necessary for

accurate taxonomic assignment of metagenomic and metataxonomic sequence data in the

future. Our re-examination of 16S rRNA gene surveys from marine sediment shows that this

phylum is surprisingly abundant and globally distributed, and therefore likely an

unrecognised but significant player in geochemical element cycling. Based on the genomes

and isolates obtained so far, members of the phylum are likely to be O2-tolerant, protein-

fermenting anaerobes, but further cultivation-dependent and independent studies will be

necessary to corroborate these initial conclusions.

Accession Numbers

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Whole Genome Shotgun projects have been deposited at DDBJ/ENA/GenBank under the

accession numbers MWJR00000000 (AABM5.125.24) and MWJS00000000 (AABM5.25.91).

The versions described in this paper are versions MWJR01000000 and MWJS01000000.

Table Caption

Table 1

Summary of genome statistics for genomes belonging to Calditrichaeota examined in this

study. Completeness statistics were generated using CheckM (Parks et al., 2015).

Figure Captions

Figure 1

Phylogenetic trees showing the candidate phylum Calditrichaeota. (A) PHYML maximum

likelihood tree based on 16S rRNA gene sequences. 100X bootstrapped - branch labels show

bootstrap percentages. Green-labeled nodes indicate sequences obtained from metagenome-

derived genome bins and single-cell genomes. (B) IQ-TREE phylogenetic tree based on a

concatenated alignment of 17 single-copy orthologous protein sequences taken from

sequenced genomes (preprotein translocase subunit SecY, 30S ribosomal proteins S13, S7,

S12, S10, S19, S3, S8, 50S ribosomal proteins L1, L2, L3, L5, L6, L11, L14, L16, and L27). The

tree is 1000X bootstrapped with branch labels showing bootstrap percentages. Numbers in

each grouped clade show the number of genomes within that clade. Labels in blue indicate

incomplete genomes without a gene encoding 16S rRNA. Currently genome sequences are not

available for Caldithrix palaeochoryensis and Calorithrix insularis and these species are

therefore not included in the tree. All sequences in red clades or with red-coloured text are

75% or more identical to the Caldithrix abyssi sequence. Clades coloured pink contain some

sequences greater than 75% identity and some sequences less than 75% identity (the number

of sequences >75% identity are shown coloured red in brackets). Sequences marked with

black-coloured text are less than 75% identical to Caldithrix abyssi.

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Figure 2

Analysis of published 16S rRNA amplicon datasets from marine and brackish sediment. (A)

World map showing sampling locations and Calditrichaeota percentage for the sample

(depth) with the highest value. At some locations, several independent cores were analysed –

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for exact coordinates see Supplemental Table ST2. (B) Calditrichaeota percentage plotted

against depth below seafloor for all datasets shown in A.

Figure 3

Protein degradation and uptake enzymes inferred from genomes in this study. Coloured

shapes show predicted proteins or protein complexes. Numbers adjacent to each protein

show the number of genomes (out of seven total genomes analysed) with genes encoding the

predicted protein or protein complex. Yellow indicates serine peptidases, green indicates

metallo peptidases, and red indicates cysteine peptidases. Orange indicates amino acid

transporters, turquoise indicates oligopeptide transporters, and blue indicates ABC

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transporters. “Sec” indicates the Sec secretion system. See supplemental tables ST4 and ST6

for details.

Acknowledgments

All sequencing was performed by the National Genomics Infrastructure sequencing platforms

at the Science for Life Laboratory at Uppsala University, a national infrastructure supported

by the Swedish Research Council (VR-RFI) and the Knut and Alice Wallenberg Foundation.

The work was supported by the Danish National Research Foundation, by grants of the

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Swedish Research Council and the Swedish Foundation for Strategic Research (grant no. 621-

2009-4813 and SSF-FFL5, respectively, both awarded to TJGE), and by grants from the

European Union Seventh Framework Program: an ERC Advanced Grant, MICROENERGY

[project no. 294200], an ERC Starting grant, PUZZLE_CELL [project no. 310039 to TJGE], and a

Marie Curie International Incoming Fellowship awarded to IPGM [ATP_adapt_energy]. We

would like to thank Susanne Nielsen, Trine Bech Søgaard and Lina Juzokaite for technical

assistance in the lab.

Conflict of Interest

The authors declare no conflict of interest.

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