Sulfurimonas microaerophilic sp. nov. and Sulfurimonas diazotrophicus sp. nov.: Two Novel Nitrogen-Fixing and Hydrogen- and Sulfur-Oxidizing Chemolithoautotrophs Within the Campylobacteria Isolated from Mangrove Sediments

Abstract

Two novel marine hydrogen- and sulfur-oxidizing bacteria, designated HSL1-7^T and HSL3-1^T, were isolated from mangrove sediments from Fujian Province, China. Strain HSL1-7^T exhibited Gram-negative, rod-shaped to slightly curved morphology with polar flagellum-driven motility, whereas strain HSL3-1^T was Gram-negative, rod-shaped and non-motile. Strain HSL1-7^T and HSL3-1^T were obligate chemolithoautotrophs, capable of using molecular hydrogen and thiosulfate as an energy source, and molecular oxygen and elemental sulfur as the electron acceptors for growth. Cellular fatty acid profiles revealed similar predominant components (C_16:1ω7c, C_16:0, C_18:1ω7c, and C_14:0) in both strains. Strains HSL1-7^T and HSL3-1^T were strongly diazotrophic, as demonstrated by ¹⁵N₂ fixation when a fixed nitrogen source was absent from the growth medium. The DNA G+C contents of strains HSL1-7^T and HSL3-1^T were determined to be 36.1% and 57.3%, respectively. Based on the 16S rRNA gene sequences, strains HSL1-7^T and HSL3-1^T exhibited the highest sequence similarities with Sulfurimonas marina B2^T (98.5% and 94.45%, respectively). Notably, the 16S rRNA gene sequence similarity between strains HSL1-7^T and HSL3-1^T was 93.19%, indicating that they represent distinct species within the genus Sulfurimonas. Comparative genomic analyses revealed the presence of diverse metabolic profiles in strains HSL1-7^T and HSL3-1^T, including carbon fixation, hydrogen oxidation, sulfur oxidation, and nitrogen fixation. The combined phenotypic, chemotaxonomic, and phylogenetic evidence, including average nucleotide identity and in silico DNA-DNA hybridization values, shows that strains HSL1-7^T and HSL3-1^T represent two novel species of the genus Sulfurimonas for which the names Sulfurimonas microaerophilic sp. nov. and Sulfurimonas diazotrophicus sp. nov. are proposed, with the type strains HSL1-7^T (=MCCC 1A18899^T = KCTC 25640^T) and HSL3-1^T (=MCCC 1A18844^T), respectively.

Keywords: Sulfurimonas diazotrophicus HSL3-1T; Sulfurimonas microaerophilic HSL1-7T; chemolithoautotroph; hydrogen oxidation; nitrogen fixation; sulfur oxidation.

Figure 1

Maximum-likelihood phylogenetic tree based on the 16S rRNA gene sequences showing the relationship between strains HSL1-7^T and HSL3-1^T with other members within the genus Sulfurimonas. Species names highlighted in bold represent novel taxa discovered in this work. Bootstrap numbers (>70%) were shown with 1000 calculations. Bar, 0.05 substitutions per nucleotide position.

Figure 2

Phylogenetic reconstruction using 92 core genes was performed for strains HSL1-7^T and HSL3-1^T, along with their functional genes involved in carbon, hydrogen, sulfur, and nitrogen metabolism, in comparison with closely related species. Bootstrap values (>70%) are indicated at nodes based on 1000 replicates. Species names highlighted in bold represent novel taxa discovered in this work. Bar, 0.2 substitutions per nucleotide position. Column descriptions are as follows: Carbon pathway: ATP citrate lyase alpha and beta subunits (AclAB), 2- oxoglutarate:ferredoxin oxidoreductase (OorABCD), and pyruvate:ferredoxin oxidoreductase (PorABCD), aconite hydratase (Acn), 2-oxoglutarate oxidoreductase alpha-, beta-, gamma-, and delta- subunits (KorABGD), succinyl-CoA synthetase alpha and beta subunits (SucCD). Hydrogen pathways: [NiFe]-Hydrogenases Group I (HydAB and HyaAB), [NiFe]-Hydrogenases Group II (HupVS), [NiFe]-Hydrogenases Group IV (Hyc, Coo and Ech). Sulfur pathways: thiosulfate oxidating Sox proteins (SoxABXY₁Z₁, SoxY₂Z₂CD), sulfide:quinone oxidoreductase (Sqr), flavocytochrome c sulfide dehydrogenase (Fcc), sulfate adenylyltransferase (CysDN), adenylylsulfate kinase (CysC), phosphoadenosine phosphosulfate reductase (CysH), ferredoxin-sulfite reductase (SirA), polysulfide reductase chain A (PsrA). Nitrogen pathways: nitrogenase molybdenum-iron (alpha- and beta-chains) and iron protein (NifDKH), periplasmic nitrate reductase components (NapABGH), nitrite reductase (NirK/NirS), nitric oxide reductase subunit A/B (NorBC), nitrous-oxide reductase (NosZ), ferredoxin-nitrate reductase (NarAB), ferredoxin-nitrite reductase (NirA).

Figure 3

Determination of nitrogen fixation activity of strains HSL1-7^T and HSL3-1^T and analysis of nitrogen fixation gene clusters. (A) ¹⁵N abundance based on the ¹⁵N incorporation assay, the incubation conditions were H₂ as the sole electron donor and O₂ as the electron acceptor, Sulfurimonas xiamenensis 1-1N^T which lacked nif gene cluster served as a control. (B) Green represents nitrogen fixation genes (nifHDK), red represents nitrogenase metal cofactor biosynthesis genes (nifBSZVENUWOQXTN), purple represents transcriptional regulator genes (P-II, nifA, araC), pink represent electron transfer genes (fdx), and yellow represents transporter genes (ABC).