Genome sequence of the homoacetogenic bacterium Holophaga foetida type strain (TMBS4^T)

Strain TMBS4^T (= DSM 6591) is the type strain of the species Holophaga foetida [1], which is the type species of the monospecific genus Holophaga [1,2]. The genus Holophaga is the type genus of the family Holophagaceae [3] in the order Holophagales [3] within the class Holophagae [3]. The genus name was derived from a combination of the Neo-Greek term holos, whole, and the Greek term phagein, to eat, meaning eating it all [1]; the species epithet was derived from the Latin adjective foetidus, smelling, stinking, referring to the production of foul-smelling methanethiol and dimethylsulfide [1]. Strain TMBS4^T was originally isolated from a black anoxic freshwater mud sample from a ditch near Konstanz, Germany [4]. It was found to transfer methyl groups from methoxylated aromatic compounds to sulfide, forming methanethiol and dimethylsulfide [4]. Dimethylsulfide plays an important role in atmospheric chemistry, and is produced mainly by marine bacteria from dimethylsulfoniopropionate (reviewed in [5]). The production of dimethylsulfide from methoxylated aromatic compounds represents a unique pathway for production of this important compound. Strain TMBS4^T anaerobically degrades several aromatic compounds to acetate [1,4]. The only other cultured species within the order Holophagales is Geothrix fermentans, which is also an anaerobe but degrades small organic acids and fatty acids using Fe(III) as an electron acceptor [6]. Here we present a summary classification and a set of features for H. foetida TMBS4^T, together with the description of the genomic sequencing and annotation.

A representative genomic 16S rRNA sequence of H. foetida TMBS4^T was compared using NCBI BLAST [7,8] under default settings (e.g., considering only the high-scoring segment pairs (HSPs) from the best 250 hits) with the most recent release of the Greengenes database [9] and the relative frequencies of taxa and keywords (reduced to their stem [10]) were determined, weighted by BLAST scores. The most frequently occurring genera were Holophaga (52.9%), Geothrix (33.7%) and Acidobacterium (13.4%) (5 hits in total). Regarding the two hits to sequences from members of the species, the average identity within HSPs was 99.7%, whereas the average coverage by HSPs was 100.0%. Among all other species, the one yielding the highest score was G. fermentans (NR_036779), which corresponded to an identity of 91.6% and a HSP coverage of 97.8%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification.) The highest-scoring environmental sequence was DQ676369 ('Archaeal sediment and plankton freshwater pond suboxic freshwater-pond clone MVP-105'), which showed an identity of 97.6% and a HSP coverage of 94.9%. The most frequently occurring keywords within the labels of all environmental samples which yielded hits were 'lake' (6.2%), 'aquat' (4.6%), 'gatun, rank' (4.3%), 'soil' (3.4%) and 'microbi' (2.1%) (245 hits in total). The most frequently occurring keywords within the labels of those environmental samples which yielded hits of a higher score than the highest scoring species were 'situ' (3.3%), 'microbi' (3.0%), 'groundwat' (2.8%), 'activ' (2.5%) and 'aquif' (2.5%) (42 hits in total), all of which are keywords with biological meaning fitting the environment from which strain TMBS4^T was isolated.

Figure 1 shows the phylogenetic neighborhood of H. foetida in a 16S rRNA based tree. The sequences of the two identical 16S rRNA gene copies in the genome differ by two nucleotides from the previously published 16S rRNA sequence (X77215), which contains one ambiguous base call.

H. foetida TMBS4^T is Gram-negative, and an electron micrograph shows an inner and outer membrane [1]. Cells are rod-shaped, 1-3 μm long and 0.5-0.7 μm wide [1,4] (Figure 2). No motility was observed [1,4], although the genome is rich in genes classified under ‘cell motility’ (152 genes). Growth was observed between 10°C and 35°C with an optimum at 28-32°C [1,4]. The pH range for growth was 5.5-8.0 with 6.8-7.5 as the optimum [1,4]. The salinity range for growth was 1-15 g/l NaCl [4]. Aromatic compounds utilized by TMBS4^T include 3,4,5-trimethoxybenzoate, syringate, 5-hydroxyvanillate, phloroglucinol monomethyl ether, sinapate, ferulate, caffeate, gallate, 2,4,6-trihydroxybenzoate, pyrogallol, and phloroglucinol [1,4]. The fastest growth occurred with syringate [4]. When sulfide was present in the medium, methyl groups from aromatic compounds were used to form methanethiol and dimethylsulfide [1,4]. Strain TMBS4^T could also grow with CO or CO2 as methyl acceptors, and acetyl-CoA synthase activity was detected [26]. Growth was also observed on pyruvate [1,4].

The genome in its current assembly consists of three scaffolds with lengths of 3,443,192 bp, 677,300 bp and 6,745 bp and a 63.0% G+C content (Table 3 and Figure 3). Of the 3,672 predicted genes, 3,615 were protein-coding genes, and 57 RNAs; 76 pseudogenes were also identified. The majority of the protein-coding genes (74.3%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

H. foetida is known to utilize aromatic compounds through the phloroglucinol pathway, producing three molecules of acetate from the benzene ring. It is also capable of growing on methoxylated aromatic compounds, transferring methyl groups to sulfide or CO2 to produce dimethylsulfide or acetyl-CoA. Genes have not been identified for the enzymes of the phloroglucinol pathway with one exception: the transhydroxylase that converts pyrogallol to phloroglucinol, which has been identified in Pelobacter acidigallici [43]. This enzyme has two subunits, and genes with high similarity to these subunits are found in H. foetida. HolfoDRAFT_0037 and HolfoDRAFT_0041 have 74% and 88% similarity to the large subunit, while HolfoDRAFT_0036, HolfoDRAFT_0040, and HolfoDRAFT_0058 have 63%, 73%, and 65% similarity to the small subunit.

H. foetida likely gains energy from the conversion of acetyl-CoA produced from aromatic compounds, pyruvate, and methyl groups from methoxylated aromatic compounds to acetate. Within the genome, there are two phosphotransacetylase genes (HolfoDRAFT_0402, HolfoDRAFT_1130) and two acetate kinase genes (HolfoDRAFT_1418, HolfoDRAFT_3547). Several candidates for pyruvate:ferredoxin oxidoreductase were found in the genome. This enzyme would produce acetyl-CoA that can be used to produce ATP and acetate. H. foetida can combine methyl groups with CO or CO2 to form acetyl-CoA, and acetyl-CoA synthase activity was detected [26]. Also within the genome, there are genes for a Moorella-type CO dehydrogenase/acetyl-CoA synthase (HolfoDRAFT_1152 and HolfoDRAFT_1153) and the two subunits of the corrinoid Fe-S protein (HolfoDRAFT_1154 and HolfoDRAFT_1157).

H. foetida has potential symporters and ABC transporters for aromatic compounds. Four genes (HolfoDRAFT_0048, HolfoDRAFT_0224, HolfoDRAFT_0791, HolfoDRAFT_0858) belonging to the major facilitator superfamily have strong similarity to aromatic compound transporters of family 2.A.1.15. H. foetida has few ABC transporters for organic compounds, but it has 3 full transporters and 8 additional substrate binding proteins from family 4. Some members of this family are amino acid transporters, but one member has been found to transport protocatechuate [44].

Systems for demethylation of methoxyaromatic compounds have been identified in Acetobacterium dehalogenans [45] and Moorella thermoacetica [46]. Methyl groups are transferred first to a corrinoid protein, then to tetrahydrofolate, by two methyltransferases. The genes for two sets of enzymes from A. dehalogenans have been sequenced [47]. The corrinoid proteins belong to COG5012, the first methyltransferases belong to COG0407, and the second methyltransferases belong to COG1410. H. foetida likely uses the same type of process. It has six proteins belonging to COG5012 and 29 proteins belonging to COG0407. The only genome with more members of COG0407 is Mahella australiensis [48] with 33. Some of the COG0407 proteins are found close to corrinoid proteins in the genome sequence. H. foetida has two members of COG1410. One is adjacent to the CO dehydrogenase/acetyl-CoA synthase genes and has 61% identity to the acsE gene of M. thermoacetica. It probably transfers methyl groups from tetrahydrofolate to the corrinoid iron-sulfur protein of CO dehydrogenase/acetyl-CoA synthase. The other COG1410 gene is adjacent to a corrinoid protein and may transfer methyl groups from corrinoid proteins to tetrahydrofolate in the methoxyaromatic demethylation pathway.