Phylogenetic analysis based on 16S rRNA gene sequences
The nearly full-length 16S rRNA gene sequence of strain HH7-29T (1513 bp) has been deposited as OR856029 in the GenBank/ database. On pairwise comparison with the 16S rRNA gene sequences available in the EzBioCloud database, strain HH7-29T exhibited the highest sequence similarities to J. alkaliphilus JC303T (98.4%), followed by J. salarius ASL-1T (98.1%), J. alimentarius YKJ-13T (98.1%) and other Jeotgalibacillus members (94.6–98.0%). These values were lower than the threshold (98.7%) for separating different species (Chun et al. 2018). In the ML, NJ and ME phylogenetic trees (Figs. 1, S1 and S2), strain HH7-29T fell within a sub-cluster of the genus Jeotgalibacillus, which consisted of five Jeotgalibacillus species (J. alkaliphilus, J. salarius, J. malaysiensis, J. alimentarius and J. terrae). Within this sub-cluster, strain HH7-29T formed an internally independent branch among adjacent strains. The taxonomic identification and evolutionary tree based on the 16S rRNA gene showed that strain HH7-29T represented a potential novel species.
Genome features and comparison
The assembled genome sequencing of strain HH7-29T (JAXQNN000000000) yielded a genome of 3284775 bp in length, which was composed of 28 contigs with an N50 value of 985906 bp (Table S1). The G + C content was 43.0%, which was in the range of 39.7–43.7% for other species of Jeotgalibacillus. A total of 3337 genes were predicted in the genome, including 3265 protein-coding genes, 16 pseudo genes and 56 RNA genes (6 rRNAs, 5 ncRNAs and 45 tRNAs). 1323 (39.6%) of all predicted genes were assigned through the RAST database. The abundance of carbohydrates metabolism (15.3%), protein metabolism (13.0%) and DNA metabolism (4.7%) in strain HH7-29T fell within the range of the closely related species (Table S2). While numbers of genes associated with respiration and potassium metabolism in strain HH7-29T were higher than that in closely related species. Meanwhile, the genes encoding hydrogenase were unique to strain HH7-29T. Additionally, genomic comparisons showed that all the nine strains shared 1352 core genes in common, accounting for 37.3–42.1% of the annotated genome repertoire respectively (Fig. 2). The antiSMASH results showed that strain HH7-29T harbors two sets of gene clusters required for carotenoid biosynthesis and siderophore biosynthesis, respectively. Additionally, the genes encoding peptidases such as ATP-dependent protease La, aminopeptidase and carboxylate peptidase were detected in the genome. In line with this, strain HH7-29T had milk and casein degradation capacity (Fig. S3).
In the phylogenetic trees based on 815 single-copy orthologous clusters (Figs. 3, S4 and S5), strain HH7-29T clustered within the clade that accommodated the genus Jeotgalibacillus, in which it formed a cluster with J. malaysiensis D5T and J. salarius ASL-1T. The ANI values between strain HH7-29T and related Jeotgalibacillus species were 71.1–83.8% (Table S3). Moreover, the dDDH values between strain HH7-29T and related species ranged from 19.5 to 27.4% (Table S3). These values are significantly lower than the threshold values for prokaryotic species delineation, which are 95–96% for ANI and 70% for dDDH (Kim et al. 2014; Meier-Kolthoff et al. 2013). Furthermore, the AAI and POCP values between strain HH7-29T and related species were 66.5–88.4% and 59.8–76.6% (Table S4), which were within the range indicating the delimitation boundaries of the genus (above 65–95% and 50%, respectively) (Luo et al. 2014; Qin et al. 2014). These results confirmed that HH7-29T represents a novel species within the genus Jeotgalibacillus.
Salinity tolerance
Under high-salinity conditions, the growth of microorganisms is severely limited. Surprisingly, strain HH7-29T could grow in NaCl concentrations up to 24%, showing high salinity tolerance ability (Table 1). It has been reported that proteins of halophilic organisms have more acidic residues and lower isoelectric points (pIs) to accommodate high ionic strength (Mongodin et al. 2005). In this study, through comparison of pIs from HH7-29T, the closely related strains, hyperhalophilic bacterium Salinibacter ruber DSM 13855T, non-halophilic bacterium Escherichia coli K-12T, these Jeotgalibacillus members exhibited strongly unimodal distributions of pI values and higher ratios of acidic residues to basic residues, S. ruber too (Fig. 4). The average pI values of all proteins in HH7-29T and the closely related strains was 6.5, significantly lower than protein pIs of E. coli K-12T with 7.1 (Table S5). Through functional annotation of genes, strain HH7-29T possesses betaine/carnitine/choline transporter, betaine/proline/choline transporter and glycine betaine transporter (Table S6). As osmoprotection, compatible solutes (e.g., glycine betaine, proline) accumulation could increase to maintain cellular osmotic balance in saline conditions (Mongodin et al. 2005; Burg and Ferraris 2008). Though the genes encoding choline dehydrogenases and betaine aldehyde dehydrogenase, which are responsible for synthesizing glycine betaine from choline (Vargas et al. 2008; Wargo 2013), were lacked, HH7-29T could encode two glutamate synthases (UFB30_01585, UFB30_01590). As an organic osmolyte, glutamate could mediate a global transcription switch at genomic promoters that vary in their response to salt (Burg and Ferraris 2008). Besides, the genome contained 13 sodium/proton antiporters, five Trk system potassium uptake protein, three sodium/calcium antiporter and two potassium/proton antiporters (Table S6), which maintained high intracellular ion concentrations within cells to achieve osmotic equilibrium. Furthermore, those sodium or potassium/proton antiporters may catalyze proton accumulation coupled with sodium efflux, thereby maintaining a cytoplasmic pH well below the high external pH in strain HH7-29T, supporting cytoplasmic pH homeostasis and alkali tolerance(Ito et al. 2004; Krulwich et al. 2009). Collectively, strain HH7-29T could not only accumulate protective organic osmolytes but also increase intracellular ion concentrations to maintain its tolerance to salt, alkali and osmolarity fluctuations.
Table 1
Differential characteristics of strain HH7-29T and type strains of closely related species.
Characteristic | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
Growth range of: | | | | | | | | | |
Temperature (optimum, ℃) | 15–40℃ (32℃) | 4–40℃ (30℃)* | 10–45℃ (30–35℃) | 4–50℃ (37℃) | 10–45℃ (30–35℃) | 15–40℃ (37℃) | 4–39℃ (30℃) | 10–40℃ (33℃) | 15–40℃ (30–37℃) |
NaCl (optimum, %) | 0.5–24% (2–9%) | 0–18% (2%)* | 0–20% (3–12%) | 0–30% (10%) | 0–20% (2–5%) | 0–9% (2%) | 0–15% (2–3%) | 0–10% (2%) | 0–9% (0–1%) |
pH (optimum) | 6.0–12.0 (8.0–8.5) | 6.0–10.5 (7.0–8.0)* | 6.0–10.0 (7.0–8.0) | 6.0–11.0 (7.0–8.0) | 6.0–10.5 (7.0–8.0) | 5.5–9.0 (8.5) | 6.0–10.5 (7.0–8.0)† | 6.0–9.5 (7.5) | 5.5–10.5 (8.0–8.5) |
Oxidase | – | + | + | + | + | – | – | – | + |
Hydrolysis of: | | | | | | | | | |
Gelatin | – | – | + | W | – | + | – | + | – |
Starch | – | – | – | + | + | – | + | – | + |
Tween 80 | + | W | + | – | – | ND | – | – | – |
Utilization of: | | | | | | | | | |
D-glucose | – | + | + | + | + | ND | + | + | – |
Maltose | + | + | + | – | – | ND | + | + | – |
Mannitol | – | + | + | + | + | – | + | – | + |
Enzyme activity (API ZYM): | | | | | | | | | |
Esterase (C4) | – | + | +* | + | + | + | –* | + | W |
Lipase esterase (C8) | + | + | +* | + | – | + | W* | ND | W |
Naphthol-AS-BI-phosphohydrolase | – | – | –* | + | + | – | W* | ND | – |
Trypsin | + | – | –* | + | – | + | –* | ND | – |
α-Chymotrypsin | + | – | –* | + | – | + | –* | ND | + |
β-Galactosidase | – | – | +* | – | – | + | +* | + | – |
α-Glucosidase | – | – | +* | + | – | + | –* | ND | + |
Reduction of: | | | | | | | | | |
Nitrates to nitrites | + | – | + | + | – | ND | + | – | – |
DNA G + C content (%) | 43.0 | 41.6* | 43.1 | 42.7 | 42.6 | 43.7 | 41.1 | 41.6 | 39.7 |
Strains: 1, Jeotgalibacillus haloalkaliphilus HH7-29T (this study); 2, Jeotgalibacillus salarius KCTC 13257T (this study); 3, Jeotgalibacillus alimentarius YKJ-13T; 4, Jeotgalibacillus malaysiensis D5T; 5, Jeotgalibacillus terrae DSM 22174T; 6, Jeotgalibacillus aurantiacus T12T; 7, Jeotgalibacillus campisalis SF-57T; 8, Jeotgalibacillus proteolyticus 22-7T; 9, Jeotgalibacillus soli P9T. |
Data for columns 3–9 taken from Yoon et al. (2001), Yaakop et al. (2015), Chen et al. (2010), Jiang et al. (2022), Yoon et al. (2004), Li et al. (2018), Cunha et al. (2012), respectively. +, positive; –, negative; W, weakly positive reaction; ND, no data ; *, Data from Yoon et al. 2010; †, Data from Li et al. 2018. |
Growth and morphological characteristics
The cells of strain HH7-29T were identified as Gram-stain positive, aerobic, rod-shaped (approximately 0.6–0.9 µm wide and 1.0–1.6 µm long) with polar flagella (Fig. S6). The strain was catalase-positive but oxidase-negative. Through antibiotic susceptibility testing, strain HH7-29T was susceptible to ampicillin, cephalexin, erythromycin, roxithromycin, oxytetracycline, streptomycin, kanamycin, sulfamethoxazole, sulfafurazole, norfloxacin, levofloxacin, vancomycin, polymyxin B, licomycin, chloramphenicol, but resistant to tetracycline. Detailed phenotypic characteristics were listed in the species description. The complete morphological and physiological differences between the species are presented in Table 1.
Chemotaxonomic characteristics
The major fatty acids (> 10.0% of the total fatty acids) of strain HH7-29T were iso-C15:0 (35.0%) and anteiso-C15:0 (15.4%). This profile is similar to that of the related species (except for J. campisalis), but with a rather wide range of abundance (11.0–57.1% and 15.2–53.5%, respectively). Furthermore, strain HH7-29T differs from the closely related species in terms of the proportions of certain moderate fatty acid components. For example, strain HH7-29T had significantly lower levels of alcohol-C17:0 (6.0%) compared with reference strain J. salarius KCTC 13257T (11.5%) (Table S7). The polar lipids compositions of strain HH7-29T was identified as phosphatidylglycerol (PG), diphosphatidylglycerol (DPG) and two unidentified phospholipids (PL1 and PL2) (Fig. S7). Therein, PG, DPG and PL2 were detected in J. salarius KCTC 13257T, but no PL1 (Fig. S7). Furthermore, the main respiratory quinones of strain HH7-29T were MK-7 and MK-8, in line with all other members of the Jeotgalibacillus representatives.
TAXONMIC CONCLUSION
The phenotypic, phylogenetic, genomic, physiological and chemotaxonomic features all support the hypothesis that strain HH7-29T represents a novel species of the genus Jeotgalibacillus, for which the name Jeotgalibacillus haloalkaliphilus sp. nov. is proposed here.
DESCRIPTION OF JEOTGALIBACILLUS HALOALKALIPHILUS SP. NOV.
Jeotgalibacillus haloalkaliphilus [hal.o.al.ka.li’phi.lus. Gr. masc. n. hals, salt; N.L. neut. n. alkali, alkali (indeclinable); Gr. masc. adj. philos, loving; N.L. masc. adj. haloalkaliphilus, loving briny and alkaline media]
Cells are Gram-stain positive, aerobic and rod-shaped (0.6–0.9×1.0–1.6 µm) with polar flagella. Colonies are yellow, circular, raised and approximately 2 mm in diameter on TYS agar after incubation for 2 days. Growth occurs at 15–40℃ (optimum, 32℃), in 0.5–24% NaCl (optimum, 2–9%) and at pH 6.0–12.0 (optimum, pH 8.0–8.5). The strain is catalase-positive and oxidase-negative. Casein, milk, Tweens 20, 40, 60 and 80 are hydrolysed, but starch, gelatin, cellulose and sodium alginate are not hydrolysed. In API 20NE tests, positive for nitrate reduction, urease, arginine dihydrolase, hydrolysis of aesculin, acid production from β-galactosidase and assimilation of maltose, gluconate, malic acid and citric acid, but negative for indole production, hydrolysis of gelatin, acidification of glucose and assimilation of glucose, arabinose, mannose, mannitol, N-acetylglucosamine, capric acid, adipate and phenylacetic acid. In API ZYM tests, there are positive reactions for alkaline phosphatase, esterase lipase (C8), trypsin, and α-chymotrypsin, but negative reactions for esterase (C4), lipase (C14), leucine arylamidase, valine aminopeptidase, cystine aminopeptidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactocosidase, β-glucuronidase, α-glucosidase, β-glucosinase, α-mannosidase, β-fucosidase, and N-acetyl-β-glucosaminidase. The predominant fatty acids are iso-C15:0 and anteiso-C15:0. The polar lipids consist of phosphatidylglycerol, diphosphatidylglycerol, and two unidentified phospholipids. The major menaquinones are MK-7 and MK-8.
The type strain, HH7-29T (= KCTC 43417T = MCCC 1K07541T), was isolated from the estuary of the Fenhe River into the Yellow River of Shanxi, China. The genomic DNA G + C content of the type strain is 43.0%. The NCBI GenBank accession numbers for the 16S rRNA gene sequence and the draft genome sequence are OR856029 and JAXQNN000000000, respectively.