ABSTRACT
Life in Movile Cave (Romania) relies entirely on carbon fixation by bacteria. The microbial community in the surface water of Movile Cave’s hypoxic air bells is dominated by large spherical-ovoid bacteria we identified as Thiovulum sp. (Campylobacterota). These form a separate phylogenetic cluster within the Thiovulaceae, consisting mostly of freshwater cave bacteria. We compared the closed genome of this Thiovulum to that of the marine strain Thiovulum ES, and to a genome we assembled from public data from the sulfidic Frasassi caves. The Movile and Frasassi Thiovulum were very similar, differing greatly from the marine strain. Based on their genomes, cave Thiovulum can switch between aerobic and anaerobic sulfide oxidation using O2 and NO3- as electron acceptors, respectively. NO3-, is likely reduced to NH3 via dissimilatory nitrate reduction to ammonia using periplasmic nitrate reductase (Nap) and hydroxylamine oxidoreductase. Thus, Thiovulum, is likely important to both S and N cycles in sulfidic subterranean aquatic ecosystems. Additionally, we suggest that the short peritrichous flagella-like structures typical of Thiovulum are type IV pili, for which genes were found in all Thiovulum genomes. These pili may play a role in veil formation, connecting adjacent cells and the exceptionally fast swimming of these bacteria.
INTRODUCTION
Movile Cave is located near the town of Mangalia, SE Romania (43°49’32”N, 28°33’38”E), 2.2 km inland from the Black Sea shore. It consists of a 200 m long upper dry passage that ends in a small lake allowing access to a 40 m long, partially submerged lower cave level (Fig. 1). Thick and impermeable layers of clays and loess cover the limestone in which the cave is developed, preventing input of water and nutrients from the surface (Lascu et al., 1994). Sulfidic groundwater flows constantly at the bottom of Movile Cave’s lower passages. Because of the morphology of the lower cave passages (Fig. 1) and a slight difference in water temperatures, the water near the surface is practically stagnant. Oxygen penetrates up to 1 mm of the water column, below which the water is anoxic (Riess et al., 1999).
Cave ecosystems, normally characterized by stable conditions, provide a window into subsurface microbiology (Engel, 2015). In the absence of natural light, these ecosystems are typically fueled by chemolithoautotrophy via the oxidation of reduced compounds such as H2S, Fe2+, Mn2+, NH3, CH4, and H+. Most of the microbiological studies performed in Movile Cave (summarized in (Kumaresan et al., 2014) are based on samples of microbial biofilms floating on the water surface or covering rock surfaces in the cave’s Air Bells (Fig. 1), where the atmosphere is low in O2 (7-10 %) and enriched in CO2 (2.5 %) and CH4
(1-2 %) (Sarbu, 2000). There, chemoautotrophic microorganisms, living at the water surface, oxidize reduced chemical compounds such as H2S, CH4 and NH4+ from the thermo-mineral groundwater (Sarbu, 2000; Sarbu and Kane, 1995; Sarbu et al., 1996). Thiobacillus, Thiothrix, Thioploca, Thiomonas and Sulfurospirillum oxidize H2S using O2 or NO3- as electron acceptors (Rohwerder et al., 2003; Chen et al., 2009; Flot et al., 2014). The methanotrophs Methylomonas, Methylococcus and Methylocystis (Hutchens et al., 2004), Methanobacterium (Schirmack et al., 2014) and Methanosarcina (Ganzert et al., 2014) are also found in the cave, alongside other methylothrophs such as Methylotenera, Methylophilus and Methylovorus (Rohwerder et al., 2003; Chen et al., 2009). Chen et al. (2009) further identified in this cave ammonia and nitrite oxidizers from the genera Nitrospira and Nitrotoga.
In the lower level of Movile Cave, directly below the water surface (not deeper than 2-3 mm) we observed a loose floating veil resembling a slow-moving white cloud (Fig. 2 and Supplementary video 1). Using genetic and microscopic analysis, we concluded that this underwater agglomeration of bacteria is dominated by a species of the genus Thiovulum (Fig. 1, S1 and results).
Thiovulum is a large bacterium, typically < 25 μm in diameter (Robertson et al., 2015) but can reach 45 μm (Sylvestre et al., 2021). It is a sulfur-oxidizing chemolithoautotrophic bacteria (Wirsen and Jannasch, 1978) with an extremely fast motility (Garcia-Pichel, 1989; Thar and Fenchel, 2001). Thiovulum is known to form a veil close to surfaces (Petroff et al., 2015; Robertson et al., 2015), to which it can attach through a secreted stalk (De Boer et al., 1961). It is normally located close to the oxic-anoxic interface near sediments or microbial mats (Marshall et al., 2012; Robertson et al., 2015; Jorgensen and Revsbech, 1983) where the 2D organization of the veil and the rapid movements of the cells’ flagella produce a convective transport of O2 (Fenchel and Glud, 1998).
To the best of our knowledge, this is the first description of fully planktonic Thiovulum swarms/veils at distance from any solid surface. Here we provide further morphological and genomic information on this bacterium, offering new insights into its metabolic properties and raising novel questions.
MATERIALS AND METHODS
A detailed description of the methods is provided in the supplementary material.
Replicate samples of water were collected into sterile containers from the surface of the small sulfidic lake and from the Air Bells in the lower section of Movile Cave (Fig. 1). Unpreserved 50 ml water samples were immediately brought to the laboratory and inspected using optical microscopy. Samples for DNA/RNA analysis were preserved in ethanol (final concentration of 50 %). Additionally, samples were preserved with formaldehyde (final concentration of 4 %) for cell enumeration. Samples for electron microscopy were fixed with 2.7 % glutaraldehyde in phosphate buffered saline (1× PBS). Samples for DNA extraction were collected in July 2019 whereas samples for RNA extraction were collected in August 2021.
Electron microscopy and elemental analysis
Fixed, dehydrated, and epoxy-embedded samples were sliced (100 nm thickness), stained with lead citrate and uranyl acetate (Hayat, 2001 and analyzed with Jeol JEM transmission electron microscope (Jeol, Japan). Samples for scanning electron microscopy (SEM) were sputtered with gold and examined on a JEOL JSM 5510 LV microscope (Jeol, Japan). Energy-dispersive X-ray spectroscopy (EDX) analysis was performed with an EDX analyzer (Oxford Instruments, Abingdon, UK) and with the INCA 300 software.
DNA and RNA Extraction
Genomic DNA was extracted using a modified version of the Omega BioTek Universal Metagenomics kit protocol (OMEGA Bio-Tek, GA, USA) (see supplementary material) from samples filtered on with a 0.2 μm Isopore membrane filter (Millipore Sigma, MA, USA).
For RNA extraction, total nucleic acids were extracted from polycarbonate filters (Millipore, 0.2 μm pore size) following Nercessian et al. (2005) with minor modifications (see supplementary material. DNA was digested by two sequential treatments with the TurboDNAfree Kit (Invitrogen ThermoFisher Scientific, Dreieich, Germany) following the manufacturer’s instructions. DNA removal was evaluated using a PCR reaction for 16S rRNA gene. First strand cDNA was then generated using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosciences, ThermoFisher scientific), and was sent for sequencing at the Core Genomic Facility at RUSH university, Chicago, IL, USA.
16S rRNA gene amplicon sequencing and processing
PCR reactions were performed in triplicates targeting the V3-V4 region of the 16S rRNA gene, using the V3 forward primer S-D-Bact-0341-b-S-17, 5’-CCTACGGGNGGCWGCAG-3 (Herlemann et al., 2011), and the V4 reverse primer S-D-Bact-0785-a-A-21, 5’-GACTACHVGGGTATCTAATCC-3 (Muyzer et al., 1993), resulting in fragments of ∼430 bp. The primers were dual barcoded in a way compatible with Illumina sequencing platforms (as described in Caporaso et al. (2011).
Composite samples were paired-end sequenced at the Vrije Universiteit Amsterdam Medical Center (Amsterdam, The Netherlands) on an Illumina MiSeq Sequencer. The paired sequences were dereplicated using the dedupe tool of the BBTools package (sourceforge.net/projects/bbmap/) aligned and annotated using the SINA aligner (Pruesse et al., 2012) against the SILVA SSU database (v 138.1) (Quast et al., 2013)
A maximum-likelihood phylogenetic tree was calculated including only long 16S rRNA sequences, using FastTree 2 (Price et al., 2010) using all Thiovulum sequences in the SILVA database (n=71), three 16S rRNA sequences obtained from the assembled genome of the Movile Cave Thiovulum (see below) and sequences of different Sulfurimonas species as an outgroup. A second tree included amplicon sequences as well. For the sake of legibility, the 908 Thiovulum sequence variants obtained were clustered at 97 % similarity using CD-HIT-EST (Huang et al., 2010), resulting in 50 clusters.
To obtain information on relative Thiovulum abundance, the raw short-read libraries (metagenomic) were analyzed with phyloFlash (V 3.3; (Gruber-Vodicka et al., 2020)).
Shotgun sequencing (Illumina and Oxford Nanopore)
Shotgun sequencing was accomplished using both Illumina and Oxford Nanopore sequencing technologies. For Illumina sequencing, 1 ng of genomic DNA from each sample was converted to whole-genome sequencing libraries using the Nextera XT sequencing reagents according to the manufacturer’s instructions (Illumina, San Deigo CA).
A first pass of Oxford Nanopore sequences was obtained using the SQK LSK109 ligation library synthesis reagents on a Rev 9.4 nanopore flow cell with the GridION X5 MK1 sequencing platform, resulting in a total of 131.8 Mbp of reads with a N50 of 1.3 kbp.
Additionally, sequencing was performed on several cellular aggregates that were confirmed microscopically to contain Thiovulum cells. The cell aggregates were lysed by freeze thawing and further, following the manufacturer’s instructions, as part of the DNA amplification process using the Repli-G single cell amplification kit (Qiagene, Hilden, Germany). Libraries for Nanopore sequencing were prepared using the LSK-108 kit following the manufacturer’s protocol but skipping the size selection step. The prepared libraries were loaded on MIN106 R9 flow cells, generating a total of 5.7 Gbp of reads with a length N50 of about 3.7 kbp. Basecalling for all Oxford Nanopore reads were done using Guppy 4.0.11.
cDNA sequencing
cDNA was sheared with the Rapid Shear gDNA shearing kit (Triangle Biotechnology, Durham, NC, USA) and used in the Swift 1S protocol (Accel-NGS 1S Plus kit, Swift Biosciences, Ann Arbor, Mi, USA) with 6 cycles of PCR during indexing. Following library prep, all libraries were pooled in equal volume by combining 2 μl of each library for a final bead clean up with 0.85X AmpPure beads (Beck-man Coulter Life Sciences, Indianapolis, IN, USA). This QC pool was then sequenced on an Illumina MiniSeq MO flow cell. The resulting index distribution was used to re-pool the libraries for an Illumina SP flow cell sequencing run with sample LR1 pooled at maximum volume available.
All sequencing data generated in this study were deposited in NCBI Sequence Read Archive under accession number PRJNA673084.
Metagenomic data analysis
Nanopore reads were assembled using Flye 2.8.1-b1676 (Kolmogorov et al., 2019) with default parameters, further manually processed using Bandage (Wick et al., 2015) following a final polishing step was performed with unicycler-polish from Unicycler v0.4.9b (Wick et al., 2017) using the complete set of Illumina reads (for a total depth of coverage of 12X of the genome) and the subset of Nanopore reads longer than 5 kb (ca. 50X). Polishing consisted of two cycles of pilon 1.23 (Walker et al., 2014), one cycle of racon 0.5.0 (Vaser et al., 2017) followed by FreeBase (Garrison and Marth, 2012), then 30 additional cycles of short-read polishing using pilon 1.23, after which the assembly reached its best ALE score (Clark et al., 2013).
The completeness of the Thiovulum genome obtained was assessed using CheckM (Parks et al., 2015) and its continuity using the unicycler-check module in Unicycler v0.4.9b. Annotation was performed using Prokka (Seemann, 2014), DRAM (Shaffer et al., 2020), KEGG (Kanehisa et al., 2016), EggNOG 5.0 (Huerta-Cepas et al., 2019), PATRIC (Davis et al., 2020; Brettin et al., 2015) and RAST (Aziz et al., 2008; Overbeek et al., 2014). A COG (Tatusov et al., 2000) analysis was done using the ANVIO tool (Eren et al., 2015). OperonMapper (Taboada et al., 2018) was used to inspect the organization of genes into operons. CRISPRs where identified using CRISPR finder tool (Grissa et al., 2007). Metabolic models of the annotated genome from Movile Cave and that of Thiovulum ES were calculated using PathwayTools (V25.3) (Karp et al., 2021).
Thiovulum sp. genome assembly from public databases
All available metagenomic libraries from the Frasassi caves in Italy (accession numbers in supplementary material) were quality-trimmed using Trimmomatic (Bolger et al., 2014) and scanned for the presence of Thiovulum 16S rRNA using PhyloFlash (Gruber-Vodicka et al., 2020). Library SRR1560850 contained >170,000 Thiovulum sp. 16S rRNA and was assembled using Megahit V. 1.2.9 (Li et al., 2015), and binned using Metabat2 (Kang et al., 2015). The bins were taxonomically annotated using the GTDB-Tk tool (Chaumeil et al., 2019) with one bin annotated as Thiovulum. The phylogenetic tree generated by the GTDB-Tk tool from a single-copy marker gene multilocus alignment suggested that the Movile and Frasassi caves Thiovulum genomes were closely related, hence, both genomes were used to recruit all Thiovulum related reads from all Frasassi libraries. The obtained reads were re-assembled, binned and taxonomically annotated, as above, resulting in a Thiovulum bin with 94 % completeness, 0.41 % contamination and 25 % strain heterogeneity as evaluated using CheckM (Parks et al., 2015).
Transcriptomic analysis
The 6 libraries containing cDNA sequences (3 from Air Bell 2 and 3 from Lake Room), were quality-trimmed using trimommatic (Bolger et al., 2014) and mapped against the complete genome of the Movile cave Thiovulum sp. using Salmon version 1.6 (Patro et al., 2017). Ribosomal RNA data were removed from the mapping results and TPM (Transcripts Per Kilobase Million) values were recalculated to reflect mRNA expression. The RNA data was analyzed using the iDEP (v. 0.95) online tool (Ge et al., 2018) that provides an online graphical user interface for the DeSEQ2 (Love et al., 2014) and Limma (Ritchie et al., 2015) packages for RNAseq analysis. Differential expression was considered significant with a 2-fold difference and a false discovery rate smaller than 0.1. Taxonomic composition of the active community was obtain by analyzing the 16S rRNA gene from the transcriptomic read libraries using PhyloFlash as above (V 3.3; (Gruber-Vodicka et al., 2020)). Viral transcripts were identified using VirSorter2 (v.1.1) (Guo et al., 2021), annotated against the viral refseq database release 209 using BLAST and quantified using Salmon version 1.6 (Patro et al., 2017).
RESULTS
Field observations
A pale-white veil, with a vertical thickness of 2 to 3 mm, was observed below and adjacent to the water surface in Movile Cave (Fig. 2), resembling microbial veils described for sulfur-oxidizing bacteria (Fenchel, 1994; Garcia-Pichel, 1989). Nevertheless, in Movile Cave, the dense agglomeration of cells does not form slime or a strongly cohesive aggregation.
Microscopy
Veils similar to those seen in the Lake Room (Fig. 2A) were also observed in Air Bell 1 and even more so in Air Bell 2 (Fig 2B) where they reached the highest densities. In Air Bell 2 these veils consisted of large, spherical to ovoid, bacterial cells (Fig. 3A-B) identified as belonging to the genus Thiovulum. These cells had a diameter of 12-16 μm, contained 20-30 sulfur globules each (Fig. 3), and occurred in densities of approximately 5.5×103 cells/ml. Transmission Electron Microscope (TEM) observations showed that these large cells were Gram-negative (Fig. 3C-D), and confirmed the existence of 20-30 irregularly shaped sulfur inclusions within each of the cells. Light and TEM imaging revealed Thiovulum cell divisions along the long cell axis (Fig. 3B, C). Short peritrichous filamentous appendages (Fig. 3D) observed on the surface of the cells resemble those noticed earlier in other Thiovulum species (Wirsen and Jannasch, 1978). Scanning electron microscopy (SEM) revealed the ball-like structure of the sulfur inclusions in a series of connected Thiovulum cells (Fig. 3E). These cells were connected one to the other via multiples threads (Fig. 3F-G). Energy-dispersive X-ray (EDX) analysis (Fig. 3H-I) confirmed that the intracellular globules contained sulfur (20.9 - 26.1 %), along with elements common in organic matter such as carbon (49 - 49.2 %) and oxygen (21.1 - 24.6 %), and a few other elements in low abundance such as sodium (2.4 - 3.4 %) and phosphorus (1.2 - 2.2 %).
Phylogenetic identification and relative abundance of Thiovulum
Thiovulum was found in highest abundance (sequence frequency) in Air Bell 2 (35 %), followed by Lake Room (5 %) and submerged microbial mats (0.9 %) (see pie charts in Fig. 1). Similarly, 35 % of the amplicon sequences obtained from Air Bell 2 were annotated as Thiovulum sp. A detailed community composition based on 16S rRNA genes obtained from the metagenomic libraries is presented in Supplementary Fig. 1. An amplicon-based multi-year study on the microbial community composition in the cave will be published separately.
The 16S rRNA sequences obtained from the closed genome of the Movile Cave Thiovulum, alongside other Thiovulum sequences obtained from Movile Cave in an earlier study (Porter et al., 2009), formed a separate clade together with other cave and subsurface, freshwater, Thiovulaceae, specifically from the sulfidic Frasassi caves in Italy (Fig. 4A). This clade stems from one of two clades of marine Thiovulaceae, one of which included Thiovulum ES, for which a draft genome is available (Marshall et al., 2012). Including shorter amplicon sequences of Thiovulum from Air Bell 2 in the phylogenetic tree (Supplementary Fig. 2), highlights the diversity of these bacteria in the cave.
A phylogenetic tree constructed from a multilocus alignment of single-copy marker genes from Thiovulum ES (Marshall et al., 2012), the Thiovulum genome from Movile Cave, a metagenome assembled genome from the Frasassi caves, and all available Sulfurimonas genomes, resulted in the Frasassi and Movile Thiovulum genomes forming a separate clade (Fig. 4B). The Movile Cave genome had an overall low similarity to the marine Thiovulum (ES) genome (Marshall et al., 2012), with an average nucleotide identity (ANI) of 74.49 %, an average amino acid identity (AAI) of 58.33 % (Fig. 5A-B), and a very low sequence synteny (Fig. 5C). In contrast, The Movile and Frasassi Thiovulum genomes were highly similar with an ANI of 97 % and an AAI of 95 %. (Fig. 5D, E), as well as a high gene synteny (Fig. 5F).
Genome analysis
The assembly of metagenomic data from Movile Cave resulted in a closed circular genomic sequence classified as Thiovulum sp. with a genome length of 1.75 Mbp (coverage X330) and a GC content of 28.4 %. Genome completeness was estimated using CheckM (Parks et al., 2015) at 93 %. Using a Campylobacteraceae specific set of marker genes did not improve the completeness prediction, however, in absence of sufficient reference genomes for this genus, this value likely represents the full set of marker genes for Thiovulum. CheckM estimated a contamination of 0 % and a strain heterogeneity of 0, suggesting the Thiovulum genome assembly does not contain any contaminating sequences from additional distant or closely related organisms.
The genome was analyzed using different tools with the results summarized in Supplementary Dataset 1. Genes discussed further on are addressed using the notation G2Y-n, where n refers to an incremental number. This notation is used by PathwayTools (Karp et al., 2021) and match the supplementary metabolic model provided (Supplementary Figures 3,4).
The genome contains 1804 coding sequences, of which 1534 could be annotated and 270 remain hypothetical proteins, 36 tRNAs genes, 3 rRNA operons and 9 CRISPR arrays in which 5 Type III Cas genes were identified (G2Y-562:567), comprising a total of 77 repeats. The same annotation conducted de novo on the Thiovulum ES genomes suggests, based on COGs (Clusters of Orthologs genes), that the Movile, ES and Frasassi strains share 879 core genes (Fig. 5G and Supplementary Dataset 2). The Movile strain further shares 33 and 777 genes with the ES and Frasassi strains, respectively. The Frasassi and ES strains had 26 common genes in addition to the core genome. The Movile, ES, and Frasassi genomes further contained 145, 1201, and 207 individual genes, respectively.
Carbon metabolism
Similar to Thiovulum ES, all genes required for C fixation via the reductive TCA cycle could be identified. in the Thiovulum genomes from Movile Cave and from Frasassi. The oxidative TCA cycle is complete as well in both the Thiovulum species with the citrate synthase gene (EC 2.3.3.1) replaced by ATP-citrate lyase (EC 2.3.3.8; GDY-1367,1367 alpha and beta subunits, respectively) (Fig. 6, Supplementary Figs 3,4, Supplementary Dataset 1).
Sulfur metabolism
All annotation approaches (Supplementary Dataset 1) revealed only few genes involved in dissimilatory sulfur cycling, including two copies of the sulfide:quinone oxidoredutase (G2Y-583, G2Y-1704) that oxidizes sulfide to polysulfide, and the polysulfide reductase gene nrfD (G2Y-67) that carries out the reverse process. nrfD was found in a 3-gene potential operon together with the large subunit of the assimilatory nitrate reductase (narB, G2Y-68) and the ttrB; tetrathionate reductase subunit B (G2Y-66). Two rhodanese sulfur transferase proteins (G2Y-815, G2Y-816) were identified in a 6-gene operon containing two other subunits of a nitrate reductase (narH: G2Y-813 and narG: G2Y-814). Among the two other genes in this operon, one is related to cytochrome C (G2Y-811) and the other could not be annotated (G2Y-810). The tauE sulfite exporter was identified (G2Y-644) as part of a 5-gene operon involved in the transport of molybdate (G2Y-641-643,645 modCABD, respectively). Sulfite dehydrogenase, dissimilatory sulfite reductase (dsrAB), the sox genes or adenylyl sulfate reductase (aprAB) that carry out the sulfide oxidation to SO 42- could not be found by any of the annotation tools nor by manual BLAST against all sequences available for each of those protein in the UniProt database.
Nitrogen metabolism
In addition to the membrane-bound nitrate reductases (nar, G2Y-813,814) found also in Thiovulum ES, the Movile and Frasassi cave Thiovulum possesses also periplasmatic nitrate reductases encoded by the nap genes encoded in one operon (G2Y-1099-G2Y-1099, napAGHB_F). The hypothetical protein encoded in this operon (G2Y-1098) is likely part of the napF gene (G2Y-1099) as seen by BLAST analysis in other Campylobacteraceae. Additionally, the gene for hydroxylamine dehydrogenase, which is often encountered in genomes from Campylobacterota (Haase et al., 2017), formerly referred to as Epsilonproteobacteria (Waite et al., 2019), was also identified (G2Y-1392) in a 3-gene operon with two unannotated hypothetical genes (G2Y-1390, G2Y-1391).
Chemotaxis and motility
As Thiovulum sp. is a highly motile bacterium, we inspected motility and chemotaxis genes. All genes necessary for flagellar assembly were found in the Movile and Frasassi strains, similarly to Thiovulum ES. The chemotaxis genes cheV, cheA, cheW, cheD (G2Y-470:472 cheVAW, G2Y-741 CheD) and cheY (G2Y-1156) were identified as well as additional cheY-like domains (G2Y-6,20,151,182,251,389,582,712,973,1116,1130,1156’,1344,1486,1516). The cetA and cetB (G2Y-174:176 cetABB’) energy taxis genes and the parallel to the Escherichia coli aerotaxis (aer) (G2Y-1557) gene were also identified. The cheX gene, which was not found in the genome of Thiovulum ES, was identified in the Movile Cave Thiovulum. Gene cheB was reported missing in Thiovulum ES, was identified in the Movile strain (G2Y-1843) but also in Thiovulum ES upon COG reannotation. Additionally, 9 methyl-accepting chemotaxis proteins were identified (G2Y-84,85,181,721,740,1225,1487,1557,1836).
In addition to flagella genes, (G2Y-3, fliC; G2Y-45, flgA; G2Y-48, fliC; G2Y-184, flhB; G2Y-302, motB; G2Y-336, flgK; G2Y-338, flgM; G2Y-350, fliN; G2Y-367, fliF; G2Y-442, flhA; G2Y-569, flgH; G2Y-656, fliG; G2Y-666, lag; G2Y-781, fliN; G2Y-790, fliI; G2Y-928, flgE; G2Y-1052, fliS; G2Y-1053, fliD; G2Y-1054, lag; G2Y-1107, flgE; G2Y-1108, flgD; G2Y-1122, flgB; G2Y-1199, fliH; G2Y-1218, fliM; G2Y-1222, flhF; G2Y-1250, fliR; G2Y-1258, flgI; G2Y-1331, fliL; G2Y-1458, motA; G2Y-1522, flgG; G2Y-1568, fliQ; G2Y-1728, flgF; G2Y-1801, fliE; G2Y-1802, flgC) the pilA, pilE, pilT, pilN, pulO, fimV genes responsible for the formation and retraction of type IV pili were identified.
Gene expression
Samples from Air Bell 2 and from the Lake Room were collected for RNA analysis to confirm that Thiovulum is active in Movile Cave. 16S rRNA of Thiovulum dominated all samples, making up more than 94 % of the active community (Figure S1) even though Thiovulum DNA was rare in the Lake Room in our previous samples. Despite the similarity in abundance, the gene expression profiles differ significantly between the two sites (Fig. 7). In both the heatmap (Fig. 7A) and the principal component analysis (Fig. 7B), the samples from the different environments clustered separately, with clear clusters of genes differently expressed in the two cave compartments. Differential expression analysis (Fig. 7C; Supplementary Dataset 3) revealed that 222 genes were more expressed in the Lake Room compared to Air Bell 2, while the opposite comparison resulted in 42 genes. Over half of the genes more expressed in the Lake Room encoded for hypothetical proteins to which no function could be assigned. Retron-type reverse transcriptases were the most dominant group of genes (n=15) also exhibiting some of the highest transcription level with TPM values up to 19,000. Genes over expressed in samples from Air Bell 2 were related to energy generation including cytochromes c and b as well as F-type ATP synthase. The entire gene expression data is available in Supplementary Dataset 1 and is additionally depicted in Fig. 6 next to the displayed genes or functions.
DISCUSSION
In Movile Cave, the oxidation of reduced compounds such as H2S, CH4, and NH4+ is the only primary energy source. Thiovulum, a large sulfur oxidizer, often found in close proximity to sediments, microbial mats or surfaces (Marshall et al., 2012; Jorgensen and Revsbech, 1983; Gros, 2017), is part of the Movile chemoautotrophic microbial community, involved in in situ carbon fixation, that represents the base of the cave’s food web that supports an abundant and diverse invertebrate community (Sarbu, 2000; Brad et al., 2021). These Thiovulum cells, exceeding 15 μm in diameter, are larger than most known sulfur-oxidizers, belonging to the group of giant sulfur bacteria (Ionescu and Bizic, 2019). Here we investigated the morphological, phylogenetic, and genomic aspects of a fully planktonic Thiovulum sp.. We further compared its genome to that of the sole other existing genome of Thiovulum, strain ES. The latter, originating from a phototrophic marine mat, was reannotated for the purpose of this comparison to account for new available information, 8 years after its original publication. The main aspects of this comparison are depicted in Fig. 6 and in more details in Supplementary Fig. 3 and 4. The close phylogenetic relationship of the Movile Cave Thiovulum 16S rRNA gene with sequences from the Frasassi caves prompted us to recover a Thiovulum genome from publicly available data. The obtained MAG was added to the discussed comparison.
Hypoxic Air Bell 2 vs. oxic Lake Room
Thiovulum is not typically dominant in microscopy-or DNA based observations from the Lake Room. Yet, its rRNA gene dominated over 94 % of the transcriptomic data, similar to its presence in the RNA samples from the hypoxic Air Bell 2. Nevertheless, it is known that community profiles obtained from DNA representing pseudo-abundance, and those from RNA, representing degree of activity, can substantially differ one from the other (e.g. Shu et al., 2019; Bižic-Ionescu et al., 2018). The presence and high activity of Thiovulum at the surface of both the Lake Room and Air Bell 2, environments that differ significantly in the overlaying atmosphere, points to the metabolic flexibility of cave-dwelling Thiovulum strains and perhaps of the entire genus.
While the two expression profiles differed significantly, it is evident (Fig. 6) that most genes recognizable as involved in cell metabolism had higher expression levels in Air Bell 2, though not all at statistically significant levels (Supplementary Dataset 1). More than half of the genes overexpressed by Thiovulum in the Lake Room could not be assigned any function making it impossible to understand it’s specific metabolic activity in that compartment of the cave. However the high expression of retron-type reverse transcriptase and Type II restriction enzymes in the Lake Room can be indicative of an ongoing phage infection (Millman et al., 2020; Pingoud et al., 2014), which may explain the reduced metabolic activity and elevated expression of defense systems, though only one CRISPR associate gene was overexpressed. Quantification of viral transcripts showed an overall higher expression in the Lake Room (Fig. S6), however, at this stage it is not possible to directly connect these transcripts to Thiovulum.
Phylogeny
Our phylogenetic analysis of all available Thiovulum spp. 16S rRNA sequences revealed two main clades of marine origin with no clear physical or biogeochemical basis for the separation. All sequences obtained from sulfidic caves, or subsurface environments (e.g., a drinking water well) formed a subclade in one of these two clades. This evolutionary transition from a marine environment towards a freshwater one likely accounts for the differences in sequence and function between Thiovulum ES, found in a phototrophic mat in a marine environment, and the Movile and Frasassi cave Thiovulum. This hypothesis should be further validated as more genomes of Thiovulum will become available.
Sulfur and nitrogen metabolism
Thiovulum presents several interesting features, such as being one of the fastest bacterial swimmers and being able to form large veils consisting of interconnected cells. Many sulfur-oxidizing microorganisms including species of Thiovulum form veils by means of what appear to be mucous threads. These threads are used by the cells to attach to solid surfaces (Fauré-Fremiet and Rouiller, 1958; Fenchel, 1994; Thar and Fenchel, 2001; De Boer et al., 1961; Wirsen and Jannasch, 1978; Robertson et al., 2015). In marine settings, such veils keep cells above sediments (Karavaiko et al., 2006) at the oxic-anoxic interface where the optimal concentration of O2 and H2S can be found.
SEM analyses indicated that the cells in the dense agglomeration in Movile Cave are at least partially interconnected. It has been hypothesized that the coordinated movement of Thiovulum cells generates convective transport of H2S or O2 to the cells (Petroff et al., 2015; Fenchel and Glud, 1998). The fully planktonic localization of the cells in Air Bell 2 means that Thiovulum here cannot use surfaces to place itself at the oxic-anoxic interphase. O2 in the Lake Room was shown to penetrate only the upper 1 mm of the water (Riess et al., 1999), and this is likely similar in the hypoxic Air Bell 2.
Thiovulum is a sulfide oxidizer as evidenced by the generation and accumulation of sulfur inclusions. The amount and type of sulfur inclusions in cells is influenced by the concentrations of H2S and O2 in the environment. Typically, cells store elemental sulfur when H2S is abundant in the environment, and later use the intracellular reserves of sulfur when the sulfide source in the environment is depleted (De Boer et al., 1961). Sulfur inclusions were also shown to form when the supply of O2 is limited and as a result the sulfur cannot be entirely oxidized to soluble sulfite, thiosulfate, or sulfate. Complete depletion of sulfur inclusions from cells is not likely in Movile Cave where abundant H2S is available (245 μM (Flot et al., 2014)) continuously and where O2 is scarce in most habitats, and specifically in Air Bell 2 (Sarbu et al., 1996). The analysis of the Movile Cave, ES, and Frasassi strains genomes identified the SQR gene responsible for the oxidation of sulfide to elemental sulfur. Nevertheless, the genes required for further oxidizing elemental sulfur to sulfate, via either of the known mechanisms, were not found. An exception to this is the possible oxidation of sulfite to sulfate via the intermediate adenylyl sulfate by Thiovulum ES, for which the gene encoding the sulfate adenylyl transferase was originally found (Marshall et al., 2012), yet, according to our re-annotation the necessary adenylyl-sulfate reductase genes apr (EC1.8.4.9) or aprA (EC1.8.99.2) are missing.
Marshall et al. (2012) proposed that Thiovulum undergoes frequent (daily) oxic/anoxic cycles that prevent continuous accumulation of elemental sulfur in the cell. We advance three additional options by which the Movile Cave and likely the Frasassi caves Thiovulum, may avoid sulfur accumulation. First, the presence of a polysulfide reductase (nrfD) suggests that the cells can reduce polysulfide back to sulfide (Fig. 6). Second, the identification of different rhodanese genes, known to be involved in thiosulfate and S0 conversion to sulfite (Poser et al., 2014), and of a sulfite exporter (tauE) in the Movile Cave strain, suggests that Thiovulum may be able oxidize elemental sulfur to sulfite and transport the latter out of the cell. Third, we propose that cave-dwelling Thiovulaceae are capable of dissimilatory nitrate reduction to ammonia (DNRA) using elemental sulfur (Slobodkina et al., 2017), a process already shown in Campylobacterota (e.g. Sulfurospirillum deleyianum) (Eisenmann et al., 1995). The Movile and Frasassi Thiovulum contain not only the nar (narGH) genes for nitrate reduction, but also the periplasmatic nap genes known for their higher affinity and ability to function in low nitrate concentrations (Pandey et al., 2020). Additionally, they harbor the gene for the epsilonproteobacterial hydroxylamine dehydrogenase (εhao). Hydroxylamine dehydrogenase is known from other Campylobacterota (e.g. Campylobacter fetus or Nautilia profundicola) and was shown to mediate the respiratory reduction of nitrite to ammonia (Haase et al., 2017). In line with the findings of Marshall et al. (2012), the hao gene was not found in the genome of Thiovulum ES upon re-annotation, suggesting that the hao gene may not be part of the core Thiovulum genome. Normally, Campylobacterota that utilize hydroxylamine dehydrogenase do not have formate-dependent nitrite reductase, matching the annotation of the Movile Cave Thiovulum. Campylobacterota typically use periplasmic nitrate reductase (nap) and do not have membrane-bound narGHI system (Kern and Simon, 2009; Meyer and Huber, 2014). Interestingly, Thiovulum ES has only Nar systems while the Movile and Frasassi strains have both types, suggesting that nap genes may be a later acquisition by cave-dwelling Thiovulaceae. Nevertheless, while genomic information is suggestive of the presence or absence of specific enzymes and pathways, additional experiments and gene expression data are required to determine which of the genes are utilized and under which environmental conditions.
Thus, we propose that, if O2 is available, sulfide is oxidized to elemental sulfur with oxygen as an electron acceptor (as may have been the case for part of the community at the time of sampling give the high expression of cytochrome c oxidase cbb3; Fig. 6). However, when cells are located below the O2 penetration depth, the Movile Cave Thiovulum may oxidize sulfide using NO3- as an electron acceptor, in a process of dissimilatory nitrate reduction to ammonium, as documented in other Campylobacteraceae. Our transcriptomic analysis, however, point out that at the time of sampling the nap and εhao genes were minimally expressed as compared to other sulfur and nitrogen metabolism genes (Fig. 6, Supplementary Dataset 1). Even though εhao expression was more than 3 times higher in samples from Air Bell 2 than in Lake Room, this suggests that the DNRA pathway was not highly active in the Thiovulum community. In contrast, the high expression of both copies of rhodanase genes as well sulfite exporter (tauE) suggest that elemental sulfur may have been converted to sulfite and excreted.
Cell motility and veil formation
Thiovulum sp. often forms large veils of interconnected cells. The threads connecting the cells are thought to be secreted by the antapical organelle located at the posterior side of the cell (De Boer et al., 1961; Robertson et al., 2015). Short peritrichous filaments (Fig. 3D) observed on the surface of the cells from Movile Cave resemble those noticed earlier in Thiovulum species and referred to as flagella (Wirsen and Jannasch, 1978). While all genes necessary for flagella assembly were found in the Movile Cave, Frasassi and ES Thiovulum strains, so were genes for type IV pili. Evaluating available electron microscopy images, we suggest that these ideas need to be revisited.
Our SEM images (as well as previous ones of connected Thiovulum cells) show connecting threads that are not exclusively polar and are much thinner than the stalk-like structure shown by de Boer et al. (De Boer et al., 1961). We propose that these structures are rather type IV pili, which are known, among other functions, to connect cells to surfaces or other cells (Craig et al., 2019). Alternatively, Bhattacharya et al. (2019) have shown the formation of cell-connecting nanotubes constructed using the same enzymatic machinery used for flagella assembly. However, it is not possible to determine this in absence of TEM images of connected cells showing the presence of the reduced flagellar base.
We further question the flagellar nature of the peritrichous structures around the cells. Inspecting the high-resolution electron microscopy images taken by Fauré-Fremiet and Rouiller (1958), there is no single structure visible resembling a flagellar motor. Additionally, the length of these structures based on de Boer et al. (1961) and a similar image in Robertson et al. (2015) suggest that these < 3 μm structures are too short for typical flagella (> 10 μm in length, c.f. (Renault et al., 2017)) and are closer to the 1-2 μm lengths known type for pili. Interestingly, Caulobacter crescentus swims at speeds of up to 100 μm s-1 using a single flagellum aided by multiple pili (Gao et al., 2014). In light of this hypothesis, we inspected the images of the fibrillar organelle at the antapical pole of the cells. The high-resolution images presented in Fauré-Fremiet and Rouiller (1958) and in de Boer (De Boer et al., 1961) show an area of densely packed fibrillar structures. Considering our current knowledge in flagellar motor size (ca. 20 nm) it is highly unlikely that each of these fibres is an individual flagellum, thus, potentially representing a new flagellar organization. Furthermore, none of our TEM images could reveal flagella motor-like structures on the cell. Given the high number of flagella-like structure around the cell it is logical to assume that at least some would be seen in the images taken. Petroff et al. (Petroff et al., 2015) investigated the physics behind the 2-dimensional plane assembly of Thiovulum veils and suggested it to be a direct result from the rotational movement attracting cells to each other. Nevertheless, as seen, SEM images show cells that are physically attached one to the other, suggesting several mechanisms and steps may be involved. Interestingly, type IV pili retraction can generate forces up to 150 pN known to be involved in twitching motility in bacteria (Craig et al., 2019). If coordinated, these may be part of the explanation of the swimming velocity of Thiovulum which is, at ca. 615 μm s-1, 5 to 10 times higher than that of other flagellated bacteria (Garcia-Pichel, 1989). Thus, genomic information and re-evaluation of electron microscopy data raise new questions concerning the nature of the extracellular structures on the surface of Thiovulum sp. and call for new targeted investigations into this topic.
CONCLUSIONS
Movile Cave is a ecosystem entirely depending on chemosynthesis. We showed that submerged near-surface, planktonic microbial accumulations are dominated by Thiovulum, a giant bacterium typically associated with photosynthetic microbial mats. We further showed that Thiovulum dominates the active fraction in surface waters of hypoxic and oxic compartments of the cave, suggesting metabolic flexibility
Our results highlight the existence of a clade of cave and subsurface Thiovulaceae that based on genomic information differs significantly from marine Thiovulum. The genomes of this planktonic Thiovulum strain as well as that of the highly similar Thiovulum from the Frasassi sulfidic caves suggest that these can perform dissimilatory reduction of nitrate to ammonium, when O2 is unavailable. Thus, Thiovulum may play a role in the nitrogen cycle of sulfidic caves, providing readily available ammonia to the surrounding microorganisms. The coupling of DNRA to sulfide oxidation provides a direct and more productive source of ammonium.
This investigation of three Thiovulum genomes, coupled with observations of current and previous microscopy images, questions the number of flagella the cells have, bringing forth the possibility that the cells may use type IV pili for rapid movement and cell-to-cell interactions.
The collective behavior of Thiovulum is still a puzzle and there may be more than one mechanism keeping the cells connected in clusters or in veils. Our SEM images suggest the cells are connected by thread-like structures. Petroff et al. (2015) show, on another strain, that there is no physical connection between the cells. They suggest that the swimming behavior of individual cells keeps the cells together. More research is therefore needed to understand if these different mechanisms are driven by strain variability, or by different environmental conditions.
ACKNOWLEDGEMENTS
The authors thank GESS team for logistics with sampling in the cave. We would also like to thank Pheobe Laaguiby at the University of Vermont for performing the outstanding Oxford Nanopore sequencing and Bo Barker Jørgensen, Emily Fleming, Carl Wirsen, and Tom Fenchel for their valuable suggestions that led to the improvement of the quality of this manuscript. We would also like to thank the Extreme Microbiome Project (XMP) for providing the DNA extraction reagents and methods as well as Laura Gray and Mehdi Keddache at Illumina Corp for providing partial sequencing reagents through its partnership with XMP. T. Brad was supported by a grant of Ministry of Research and Innovation, project number PN-III-P4-ID-PCCF-2016-0016 (DARKFOOD), and by EEA Grants 2014-2021, under Project contract no. 4/2019 (GROUNDWATERISK). S. M. Sarbu was supported by a grant of Ministry of Research and Innovation (UEFISCDI) projects number PN-III-P4-ID-PCE-2020-2843 (EVO-DEVO-CAVE). J.W. Aerts acknowledges the support from a grant from the User Support Programme Space Research (grant ALW-GO/13-09) of the Netherlands Organization for Scientific Research (NWO). M. Bizic was funded through the German Research Foundation (DFG) Eigene Stelle project BI 1987/2-1. The computational resources for the assembly of the Thiovulum genome were provided to J.-F. Flot by the Consortium des Équipements de Calcul Intensif (CÉCI) funded by the Fonds de la Recherche Scientifique de Belgique (F.R.S.-FNRS) under Grant No. 2.5020.11; D. Ionescu was funded through the German Research Foundation (DFG) Eigene Stelle project IO 98/3-1.