Complete genome sequence of Priestia megaterium S188, a hydrogen sulfide-degrading bacterium

Malodor generation during livestock production is a major problem in livestock farms, as it can negatively affect animals, humans, and the environment [1]. Particularly, hydrogen sulfide (H₂S), which is a colorless gas heavier than air with a rotten egg-like odor, can cause severe distress to livestock workers [2,3]. Priestia megaterium S188, originally isolated from soil, can reduce H₂S levels in manure [4]. In this study, we sequenced the complete genome of P. megaterium S188. Initially, S188 was grown in Nutrient Broth (Difco, Tucker, GA, USA) at 30°C for 24 h. The genomic DNA of strain S188 was extracted as described in a previous study [5], and its quality was checked using a spectrophotometer (UV-1601PC, Shimadzu, Kyoto, Japan). The genome of S188 was sequenced using the PacBio RSII platform (ver. 2.0; Pacific Biosciences) at Macrogen (Seoul, Korea). All the generated reads were de novo assembled using the RS HGAP Assembly (ver. 3.0) program. The assembled S188 genome was annotated using Prokka v.1.14.6, and the RAST was accessed at https://rast.nmpdr.org on May 10, 2024. BlastP (https://www.ncbi.nlm.nih.gov/blast/), UniProt (https://www.uniprot.org), ClustalOmega (https://www.ebi.ac.uk/jdispatcher/msa/clustalo), and EggNOG-mapper (http://eggnog-mapper.embl.de) were used for the annotation, alignment, and identification of proteins. KEGG (Kyoto Encyclopedia of Genes and Genomes) Mapper (https://www.kegg.jp/kegg/mapper/) was used to map the genes of strain S188 to different metabolic pathways, and the DNA plotter in Artemis (v.18.2) was used to generate the genome maps of strain S188.

The S188 genome has a total length of 5,407,472 base pair (bp), with a chromosome size of 5,278,689 bp, and a putative plasmid of 128,783bp (Fig. 1). It has a guanine-cytosine (GC) content of 37.9% and comprises 5,761 genes, of which 5,494 are coding DNA sequences (CDS), 111 miscellaneous RNA, 118 transfer RNA, 37 ribosomal RNA, and one transfer-messenger RNA (Table 1). Genes related to H₂S metabolism (i.e., assimilation/conversion) were identified using KEGG Mapper and manual curation of the reported genes associated with sulfur metabolism. Putative genes related to H₂S assimilation and conversion, including one O-acetylhomoserine (O-AH) sulfhydrylase, two cysteine synthases, five rhodanese or sulfurtransferases, and one nitrogen reductase, were identified.

Fig. 1. Genome maps of the Priestia megaterium S188 chromosome (Left) and plasmid (Right). Circles illustrate the following features from the outside to the center: (1) Coding sequences on the forward strand, (2) coding sequences on the reverse strand, (3) Miscellaneous RNA (4) Transfer RNAs (tRNAs), (5) ribosomal RNAs (rRNAs), (6) G + C content, and (7) G + C skew. The figure was generated using DNA Plotter implemented in Artemis (v.18.2). G + C, guanine + cytosine.

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The amount of H₂S released by yeast into the environment depends on the levels of sulfide (S²⁻) available [6]. Sulfide can be incorporated into sulfur-containing amino acids such as cysteine and methionine when it condenses with O-AH, a reaction catalyzed by O-AH sulfhydrylase, or with O-acetyl-L-serine, a reaction catalyzed by cysteine synthase [6,7]. Serine serves as a precursor for the biosynthesis of S-containing amino acids [6]; the expression of genes related to serine biosynthesis is lower in Saccharomyces. cerevisiae strains that produce H₂S than in non-H₂S producers. This suggests that the facilitation of S²⁻ incorporation into amino acids due to increased amounts of intracellular serine results in reduced H₂S production. [6]. In the S188 genome, genes for the assimilation of sulfide as well as the biosynthesis of serine, cysteine, and methionine were mapped using KEGG Mapper (Table 2).

In eukaryotic cells, rhodanese, a mitochondrial sulfur transferase, is part of the mitochondrial sulfide oxidation pathway involving sulfide quinone oxidoreductase, persulfide dioxygenase (PDO), and sulfite oxidase. This pathway ultimately oxidizes H₂S to thiosulfate and sulfate [8]. In some bacteria, particularly Staphylococcus aureus, naturally occurring PDO-rhodanese fusion proteins (i.e., CstB) are involved in H₂S detoxification [8,9]. Five putative sulfur transferases or rhodanese-like domain-containing proteins, which showed 18%–––27% similarity to the CstB of S. aureus, were also identified in the genome of S188 (Table 2).

The presence of nitrate reductase (Table 2) in the S188 genome indicates another possible mechanism whereby S188 can remove or reduce H₂S. H₂S removal using nitrate-reducing and sulfide-oxidizing bacteria has also been explored; herein, H₂S serves as the electron donor for the reduction of nitrate to nitrogen gas [10].

P. megaterium S188 isolated from the soil can reduce the levels of H₂S in manure and has the potential to reduce malodors in livestock farms. The involvement of the identified putative genes related to this phenotype, such as O-AH sulfhydrylase, cysteine synthase, putative sulfur transferases, rhodanese-like domain-containing proteins, and nitrate reductase, warrants further experimentation and validation. Nonetheless, the identification of these genes offers insights into the possible mechanisms by which S188, whether alone or in synergy with other nitrate-reducing, sulfur-oxidizing bacteria, reduces the levels of H₂S and would facilitate the optimization of S188 activity to achieve more efficient H₂S removal. Finally, complete cobalamin biosynthetic (cob) operon was also deteced in the genome of P. megaterium S188 (data not shown), indicating that S188 is able to synthesize vitamin B₁₂, which needs to be investigated in the future.