16S rRNA gene sequence and phylogenetic analysis
The almost complete 16S rRNA gene sequence of Z654T (1488bp) was obtained by PCR and cloning. Phylogenetic analysis of 16S rRNA gene sequences showed that the 16S rRNA gene sequence of strain Z654T was closed with H. arcticus KCTC 42129T (97.1%), and H. namhaensis KCTC 32362T (96.3%) (Kim et al., 2014). The phylogenetic trees constructed with different tree generation algorithms (Fig. 1) showed that strain Z654T was located in a phylogenetic tree composed of H. arcticus KCTC 42129T, and H. namhaensis KCTC 32362T. Strain Z654T was placed on a node that was more distantly related to the other strains. Similar tree topologies were also shown in the 16S rRNA genes based phylogenetic trees with the maximum-likelihood and maximum-parsimony algorithms. (Fig. S1 and Fig. S2), available with the online Supplementary Information). The above results support that strain Z654T was a unique new species of the genus Halocynthiibacter.
Draft Genome Sequence Analysis
The draft genome of strain Z654T was 3296250 bp and the N50 length was 1154162 bp. The longest scaffold was 1576913 bp. An average genome depth of 467× was achieved. The output report of the genomic information annotated by PGAP showed that there were 3227 genes, 3168 protein-coding genes, 39 tRNA, 3 rRNA, 3 ncRNAs, and 1 full sequence of 16S rRNA genes in the inclusions. The ANI and dDDH values for strain Z654T and its closest relative species H. arcticus KCTC 42129T were 70% and 14.6–18.2%, respectively. 95–96% of the ANI and 70% of the dDDH values are generally considered as species boundaries (Chun et al., 2018). After these calculations, strain Z654T can be classified as a new species within the genus Halocynthiibacter. Phylogenetic analysis of the genome amino acid sequences using the FastTree and IQTree method showed that strain Z654T formed a unique lineage in the Halocynthiibacter genus (Fig. 2 and Fig. S3).
Due to the emission of large amounts of organic or inorganic pollutants from human activities [Gao et al., 2017; Luo et al., 2010], mollusks such as abalone living in sediments may accumulate many pollutants from long-term exposure to fairly high levels of chemical stress (Xing et al., 1997). In particular, cadmium, arsenic, lead and mercury are heavy metals with known biological toxicity (Jiang et al., 2015). We identified certain key genes: ACR3 and ArsH associated with arsenic resistance in strain Z654T. The cluster of gene ACR3 previously was shown to confer arsenical resistance in Saccharomyces cerevisiae. The overexpression of ACR3 induced high level arsenite resistance (Wysocki et al., 1997). ArsH contributes to the mitigation of toxicity in As by mediating the reduction of ROS generated in vivo upon exposure to oxygen anions, for example by generating FMNH2 to facilitate ROS quenching activity (Páez-Espino et al., 2020).
After detailed analysis of the metabolic pathways of Z654T by the KEGG database, the results showed that the most genes were on environmental information processing, followed by genetic information processing and carbohydrate metabolism. In addition, we have comparatively analyzed the metabolic pathways of the three species, including carbohydrate metabolism, energy metabolism, lipid metabolism, nucleotide metabolism, amino acid metabolism, and metabolism of cofactors and vitamins. Three Halocynthiibacter strains' genomic analyses revealed that they had a large number of genes involved in carbohydrate metabolism, all strains have complete pathways of glycolysis (M00002), gluconeogenesis (M00003), pyruvate oxidation (M00307), citrate cycle (M00009, M00010, M00011), pentose phosphate pathway (M00004, M00006, M00007), PRPP biosynthesis (M00005), Entner-Doudoroff pathway (M00008) and several other carbohydrate metabolism (Fig. 3). Also, only strains Z654T and H. arcticus KCTC 42129T, which possess the complete d-galacturonate degradation (M00631), d-Glucuronate degradation (M00061), d-galactonate degradation (M00552) pathways, and strain H. namhaensis KCTC 32362T, which does not (Fig. 3). This gives them a strong carbohydrate utilization capacity and this property plays an important role in their gastrointestinal colonization and probiotic action (Cui et al., 2021). At the same time, Z654T has some strain specificity. Only strains Z654T possess a complete dissimilatory nitrate reduction (M00530), denitrification (M00529), ectoine biosynthesis (M00033), polyamine biosynthesis (M00134), whereas the pathway was not complete in the other two strains (Fig. 3).
BPGA was used to conduct a pan-genome analysis to find orthologous groupings among strains Z654T, H. arcticus KCTC 42129T, and H. namhaensis KCTC 32362T. A comparative analysis based on the orthologous groups of protein revealed a total of 1439 core genes shared in the three species. The percentage of core genomes per genome ranged from 35.0–45.5%. In the three genomes, the ratio of accessory genes to unique genes differed significantly (Fig. 4). Core genes contribute more to energy metabolism, folding sorting and degradation, neurodegenerative diseases, nucleotide metabolism, replication and repair, Transcription, and translation than unique and accessory genes (Fig. S7). The accessory genes and unique genes content in strain H. arcticus KCTC 42129T were higher than in several other strains (Fig. 4). Accessory and unique genes play a larger role than core genes in carbohydrate metabolism, lipid metabolism, membrane transport and xenobiotic degradation metabolism, especially cell motility (Fig. S7). It was verified that the bacterium has gliding properties, which may confer a certain nutrient-seeking behavior (Sato et al., 2021) and increase the environmental adaptability of the bacterium in the abalone viscera.
Four probable biosynthetic gene clusters were discovered in the genome of strain Z654T based on secondary metabolite analyses predicted by antiSMASH. These clusters include one RiPP-like cluster, one hserlactone cluster, one ectoine and one terpene cluster region. These synthesized secondary metabolites are considered a very promising source of novel pharmaceutical compounds (Chiriac et al., 2018).
According to reports, seaweed-associated species including marine invertebrates and bacteria are the primary hosts of enzymes that could catabolize seaweed polysaccharides. The most significant manufacturers of these enzymes are phytophagous marine gastropods, which include sea hares and abalone (Ojima et al., 2018). Different polysaccharide-degrading enzymes have so far been identified from these animals' hepatopancreas and digestive fluids, such as alginate lyase, 1,3-glucanase, mannanase, and cellulase (Ojima et al., 2018). Subsequently, we analyzed the carbohydrate activity of strain Z654T. Strain Z654T contained 108 carbohydrate-active enzymes (Fig. S4). Among these carbohydrate-active enzymes, glycosyltransferase (GTs) were the most numerous enzymes, with 65 in total. This is followed by the glycoside hydrolases (GHs) family, with 30 in total, which proves that the strain has a strong ability for polysaccharide degradation. Besides, there were two polysaccharide lyases (PLs) in strain Z654T, which were not annotated in H. arcticus KCTC 42129T and H. namhaensis KCTC 32362T. Considering that the abalone Haliotis discus hannai feeds mainly on brown algae, therefore its gut microorganisms may be involved in polysaccharide degradation processes (Nam et al., 2018). The strong polysaccharide degradation ability of this strain helps abalone to extract energy from brown algae polysaccharides and survive better.
Phenotypic Characterization
Cells of strains Z654T were Gram-stain-negative, rod-shaped and facultatively aerobic. Colonies grown on MA were round, convex, flaxen with smooth, moist edges, besides the SEM (Fig. S5) showed Z654T cells were approximately 0.2–0.8 µm in width and 0.7–3.4 µm in length. The cells were gliding on the semisolid agar. Optimal growth occurred at 30°C (range 15–37°C) and pH 7.0 (range 6.0–8.5) with 2% (w/v) NaCl (range 1.0–10.0%). No growth was observed under anaerobic conditions. The cells were positive for nitrate reduction and negative for catalase and oxidase activity. The hydrolysis of Tween 40 and starch was detected, but casein, cellulose, alginate and Tweens 20, 60, 80 were not hydrolyzed. Despite strain Z654T showed many common traits with H. arcticus KCTC 42129T and H. namhaensis KCTC 32362T including the positive reaction of esterase (C4), naphthol-AS-BI-phosphohydrolase, negative for trypsin, α-galactosidase, β-glucuronidase, α-mannosidase, gelatinase, glucose, mannitol. However, there were some differences between the strains, such as that Z654T showed positive for the esterase lipase (C8) and lipase (C14), while H. arcticus KCTC 42129T and H. namhaensis KCTC 32362T were negative for those. Further detailed characteristics determined by the API ZYM, API 20NE, API 50CHB identification systems (BioMérieux) and Biolog GEN III MicroPlates of strains are given in Table S2. Strain Z654T showed resistance to neomycin (30 µg), streptomycin (10 µg), gentamycin (10 µg), carbenicillin (100 µg), tetracycline (30 µg), kanamycin (30 µg), polymyxin B, ampicillin (10 µg), tobramycin (10 µg), penicillin (30 µg) and norfloxacin (10 µg).
Chemotaxonomic Analysis
The only isoprenoid quinone detected in strain Z654T is ubiquitin-10 (Q-10), which is the same as the description of all members of the genus Halocynthiibacter. The major cellular fatty acid of strain Z654T were C18:0 (17.0%), C18:1 ω7c 11-methyl (13.3%) and summed future 8 (C18:1 ω7c and/or C18:1 ω7c) (58.0%) (Table S1). The summed future 8 was also the main fatty acid for the other two reference strains. The main polar lipids of strain Z654T were found to include phosphatidylcholine (PC), phosphatidylglycerol (PG), two unidentified aminolipid (AL1, AL2) and five unidentified lipids (L1, L2, L3, L4, L5). PC and PG were also present in the type strains H. arcticus KCTC 42129T and H. namhaensis KCTC 32362T, and these strains both contained AL. Three phospholipids (PL1, PL2, PL3) and aminophospholipid (APL) were identified in H. namhaensis KCTC 32362T. Z654T contained a higher amount of unidentified lipids. The further detailed polar lipids images were shown in Fig. S6.
According to all these results of phenotypic, biochemical and physiological analyses, together with the phylogenetic differences, strain Z654T can be assigned to the genus Halocynthiibacter within the family Rhodobacteraceae, as representing a novel species, for which the name Halocynthiibacte halioticoli sp. nov. is proposed.
Description of Halocynthiibacte halioticoli sp. nov.
Halocynthiibacte halioticoli (ha.li.o.ti.co.li. N.L. fem. n. Haliotis, the genus name for abalone, a rock-climbing gastropod mollusk; L. neut. n. colon, colum, great gut; N.L. gen. neut. n. halioticoli, of the gut of Haliotis).
Cells are Gram-stain-negative, rod-shaped and facultatively aerobic. Colonies grown on MA were round, convex, and flaxen with smooth, moist edges. Cells are 0.2–0.8 µm in width and 0.7–3.4 µm in length. The cells can grow in 15–37°C (optimium at 30°C), pH 6.0–8.5 (optimium at pH 7.0), and NaCl concentration of 1–10% (w/v)(optimium at 2%). Starch and Tween 40 are hydrolyzed, but agar, DNA, CM-cellulose, alginate and Tweens 20, 60 and 80 are not. Cells are positive for alkaline phosphatase, leucine arylamidase, valine arylamidase, cystine arylamidase, a-chymotrypsin, acid phosphatase, β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, β-fucosidase, Voges–Proskauer reaction. Acids are produced from d-arabinose, l-arabinose, d-xylose, potassium 5-keto-gluconate. The predominant quinone was Q-10 and the major fatty acids were C18:0, C18:1 ω7c 11-methyl and summed future 8. The polar lipids were consisted of PC, PG, three unidentified aminolipids and five unidentified lipids. The DNA G + C content of strain Z654T is 54.2 mol%.
The type strain, Z654T (= MCCC 1H00503T = KCTC 92003T), was obtained from the entrail of abalone in Rongcheng, Shandong province, China. The Whole Genome Shotgun project has been deposited at GenBank under the accession JAOYFC000000000. The accession number of the 16S rRNA gene sequence is OP218735.