Phenotypic, physiological and biochemical characteristics
After incubation at 28 °C on MA for 3 days, the colonies of strain C305T were circular, smooth, convex, entire and orange. Cells of strain C305T were Gram-reaction-negative, non-motile, rod, about 0.3–0.5 μm in diameter and 0.9–1.6 μm in length (Supplementary Fig. S1). The novel isolate could be clearly distinguished from all recognized species of the genus Brumimicrobium by the growth temperature range (4–40 °C for strain C305T and B. aurantiacum N62T, 11–36 °C for B. mesophilum JCM 14063T and -2–25 °C for B. glaciale LMG 21434T) and by the tolerance to NaCl (0.5–10.0 % for strain C305T, 0.5–13.0 % for strain B. aurantiacum N62T, 0.4–7.0 % for B. mesophilum JCM 14063T and 0.5–8.0 % for B. glaciale LMG 21434T). The strain was susceptible to (μg per disc) penicillin (10 ug),cefotaxime sodium (30 ug), chloramphenicol (30 ug), vancomycin (30 ug), erythromycin (15 ug), ceftriaxone (30 ug), clarithromycin (15 ug), rifampin (5 ug) but resistant to streptomycin (10 ug), carbenicillin (100 ug), streptomycin (10 ug), kanamycin (30 ug), gentamycin (10 ug), tobramycin (10 ug), neomycin (30 ug), tetracycline (30 ug), lincomycin (2 ug), vancomycin (30 ug), norfloxacin (10 ug), nalidixic acid (30 ug), sulfamethoxydiazine (5 ug), latamoxef (30 ug), clindamycin (30 ug). Strain C305T and the three reference strains had some phenotypic characteristics in common. And, positive for peroxidase activity, which hydrolyzes gelatin, and negative for oxidase activity and H2S reaction. Despite the common features, there were some different phenotypic characteristics between strain C305T and closely related known species of the genus Brumimicrobium (Table 1 and Supplementary Table S2). Nitrate was reduced by B. aurantiacum N62T and B. glaciale LMG 21434T, but not reduced by strain C305T and B. mesophilum JCM 14063T. In contrast to its closest neighbor, B. mesophilum JCM 14063T, strain C305T could produce naphthol-AS-BI-phosphohydrolase, hydrolyzed gelatin but not starch.
Table 1 Characteristics that distinguish strain C305T from its closely related species with validly published names.
Characteristic
|
1
|
2
|
3
|
4
|
Cell size (μm)
|
0.3-0.5
0.9-1.6
|
0.3-0.4*
0.8-1.5*
|
0.3-0.5†
0.8-1.7†
|
0.3-0.4‡
1-3‡
|
Gliding motility
|
-
|
-
|
-†
|
+‡
|
Salinity (% NaCl)
|
|
|
|
|
Range
|
0.5-10.0
|
0.5-13.0*
|
0.4-7.0†
|
0.5-8.0‡
|
Optimum
|
1.5-3.0
|
2.0-3.0*
|
3.5†
|
ND‡
|
Temperature (°C)
|
|
|
|
|
Range
|
4-40
|
4-40*
|
11-36†
|
-2-25‡
|
Optimum
|
30-33
|
28-30*
|
26†
|
16-19‡
|
pH for growth
|
|
|
|
|
Range
|
6.0-9.0
|
5.5-9.0*
|
6.5-10†
|
ND‡
|
Optimum
|
8.0
|
7.0-7.5*
|
7.5†
|
ND‡
|
Nitrate reduction
|
-
|
+*
|
-†
|
+‡
|
Hydrolysis of:
|
|
|
|
|
Starch
|
-
|
-*
|
+†
|
-‡
|
CM-cellulose
|
-
|
-*
|
ND†
|
-‡
|
Tween 20
|
-
|
+*
|
ND†
|
ND‡
|
Tween 40
|
-
|
+*
|
-†
|
+‡
|
Tween 60
|
-
|
+*
|
ND†
|
ND‡
|
Tween 80
|
-
|
+*
|
-†
|
+‡
|
Enzyme activity:
|
|
|
|
|
arginine dihydrolaze
|
+
|
-
|
-
|
-
|
citrate utilization
|
+
|
-
|
-
|
-
|
tryptophan deaminase
|
+
|
-
|
-
|
-
|
arginine dihydrolaze
|
+
|
-
|
-
|
-
|
Voges–Proskauer
reaction
|
+
|
-
|
-
|
-
|
lipase (C14)
|
-
|
-
|
+
|
+
|
naphthol-AS-BI-phosphohydrolase
|
+
|
+
|
-
|
-
|
Strains: 1, strain C305T; 2, B. aurantiacum N62T; 3, B. mesophilum JCM 14063T; 4, B. glaciale LMG 21434T.
Data of strain 1were obtained from this study
Symbols: +, positive; w, weakly positive; –, negative; ND, no data.
* Data from Zhang et al. (2017)
† Data from Yang et al. (2013)
‡ Data from Yang et al. (2013)
All strains are orange colony color and can hydrolyze gelatin. All strains are use MK-6 as major quinone. All strains are positive for catalase, alkaline phosphatase alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystinearylamidase, acid phosphatase but negative for oxidase, urease, o-nitrophenyl b-D-galactopyranoside, lysine decarboxylase, ornithine decarboxylase, o-nitrophenyl b-D-galactopyranoside, a-galactosidase,b-galactosidase,b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, b-fucosidase, H2S production and indole production.
Genome Insight of Five Strains Belonging to the genus Brumimicrobium
Basic molecular studies and phylogenetic analysis
The almost complete 16S rRNA gene sequence of strain C305T determined in this study comprised 1451 nucleotides (GenBank accession number MH000430). Strain C305T shows 98.0 % 16S rRNA gene sequence similarity to the closest relative B. mesophilum JCM 14063T and 97.5 % 16S rRNA gene sequence similarity to B. salinarum KCTC 62372T, with less than 96.9 % sequence similarity to other close relatives with validly published names. The phylogenetic trees constructed based on the 16S rRNA gene sequences of type strain of the genus Brumimicrobium showed strain C305T formed a tight phylogenetic cluster within the B. mesophilum JCM 14063T (Fig. 1). The same topology is supported by the maximum-likelihood (ML)(Supplementary Fig. S2), neighbor-joining (NJ) and maximum-parsimony (MP) (Supplementary Fig. S3) trees. Phylogenetic analysis of the genome amino acid sequences using the FastTree method showed that strain C305T formed a unique lineage in the Brumimicrobium genus (Fig. 2). Draft genome sequencing of strain C305T (QFRJ00000000) yielded a genome of 3,846,876bp in length after assembly, producing 38 contigs, and the N50 value is 255,020. All contigs were larger than 526 bp, the largest being 675,880 bp. In addition to strain C305T, the genomes of four currently known strains of the genus Brumimicrobium were also analyzed (Table 2). In this regard, some basic genomic characteristics of these five strains were obtained. Among them, the longest genome sequence was B. glaciale LMG 21434T and the shortest was B. salinarum KCTC 62372T. The G+C content of strain C305T was 35.5 mol%, which was higher than the other four strains of the genus Brumimicrobium.
Table 2 Genome statistics of strain C305T and four members of the genus Brumimicrobium.
|
1
|
2
|
3
|
4
|
5
|
Genome size (bp)
|
3,846,876
|
3,423,340
|
3,882,840
|
3,413,540
|
4,271,179
|
Contigs
|
38
|
25
|
89
|
36
|
12
|
N50 length (bp)
|
255,020
|
245,212
|
202,362
|
226,131
|
627,632
|
G+C content (mol %)
|
35.5
|
33.9
|
33.6
|
34.3
|
33.8
|
Genes
|
3,205
|
2,982
|
3,410
|
3,031
|
3,515
|
Protein-coding genes
|
3,133
|
2,921
|
3,351
|
2,926
|
3,448
|
tRNA genes
|
37
|
35
|
35
|
37
|
35
|
rRNA genes
|
6
|
5
|
3
|
4
|
7
|
Other RNA genes
|
3
|
3
|
3
|
3
|
3
|
GenBank ID
|
QFRJ
00000000
|
QURB
00000000
|
PVSS
00000000
|
PJNI
00000000
|
SETE
00000000
|
The average nucleotide identity (ANI) values between strain C305T and its closely related type species were calculated using the OrthoANIu algorithm (EzGenome web service) (Yoon et al., 2017). Digital DNA-DNA hybridization (dDDH) values were calculated by the Genome-Genome Distance Calculator website (http://ggdc.dsmz.de/distcalc2.php) (Meier-Kolthoff et al., 2013). Then, the percentage of conserved proteins (POCP) was calculated according to the method of Qin et al (Qin et al., 2014), and the average amino acid identity (AAI) values were calculated by the AAI calculator (http://ekhidna2.biocenter.helsinki.fi/AAI). ANI and dDDH values (Table 3) were below the recommended thresholds for species delimitation 95% (ANI) and 70% (dDDH) (Goris et al., 2007), while POCP and AAI values (Table 3) were within the range indicating the delimitation boundaries of the genus (above 50% and 60-90%, respectively) (Qin et al., 2014). These data suggest that strain C305T should be a member of the genus Brumimicrobium.
Table 3 ANI, dDDH, POCP and AAI values (%) between strain C305T and other type strains of the genus Brumimicrobium.
Strain
|
ANI
|
dDDH
|
POCP
|
AAI
|
Brumimicrobium aurantiacum
N62T
|
75.9
|
20.9
|
71.3
|
77.9
|
Brumimicrobium mesophilum
JCM 14063T
|
78.2
|
22.6
|
69.3
|
81.7
|
Brumimicrobium salinarum
KCTC 62372T
|
74.7
|
21.3
|
69.3
|
76.1
|
Brumimicrobium glaciale
LMG 21434T
|
79.8
|
24.2
|
70.1
|
84.0
|
Strains: 1, C305T; 2, B. aurantiacum N62T; 3, B. mesophilum JCM 14063T; 4, B. salinarum KCTC 62372T; 5, B. glaciale LMG 21434T
Metabolic Pathways
The prediction of the genomic amino acid sequence of strain C305T by KEGG revealed its complete metabolic pathway, while comparing it with the four strains of the genus Brumimicrobium (Details are in Fig.3). The results of amino acid sequence annotation of strain C305T using KEGG showed that 1314 (42.4 %) genes were predicted to function, including carbohydrate metabolism (108), signaling and cellular processing (136), genetic information processing (168), amino acid metabolism (85), environmental information processing (53), cofactor and vitamin metabolism (69), glycan biosynthesis and metabolism (49), and energy metabolism (50). The annotated results showed that a large number of genes involved in carbohydrate metabolism were present in all strains of the genus Brumimicrobium, including various carbohydrate metabolic pathways such as intact glycolysis (M00002), gluconeogenesis pathway (M00003), pentose phosphate pathway (M00007), pyruvate oxidation (M00307), and PRPP biosynthesis (M00005) (Fig. 3), except for strain B. salinarum KCTC 62372T, which does not contain the complete pyruvate oxidation pathway (M00307). In addition, it is worth noting that for amino acid metabolism, none of the strains of the genus Brumimicrobium had valine/isoleucine biosynthesis (M00019), isoleucine biosynthesis (M00570), except for strain C305T. In the metabolism of cofactors and vitamins,the KEGG gene annotation revealed that all strains have a complete pathway for haemoglobin synthesis. Haemoglobin is a catalytic material with the active center of a peroxidase enzyme. It is a porphyrin-like compound that is a cofactor for haemoglobin, myoglobin, etc. and is responsible for the transfer of electrons and oxygen in living organisms. In practical applications, haemoglobin and its porphyrin derivatives are used for the color enhancement of foodstuffs, anaemia and the treatment of porphyrias. In addition, haematoxylin-based proteins such as Dye-Decolorising Peroxidase (DyP) can be used for the degradation of waste dyes. The current extraction of haemoglobin by enzymatic methods and from biological materials has disadvantages such as low yields and environmental unfriendliness. The production of haemoglobin and recombinant haemoglobin can be obtained by using microbial protein might be a good route in the future.
Prediction of Secondary Metabolites
Predictive analysis of the secondary metabolites of C305T and the remaining four strains of its genus Brumimicrobium, based on antiSMASH, identified one common cluster of possible biosynthetic genes in the genome of genus Brumimicrobium. A terpene cluster region was present in all five strains. According to recent studies, in addition to the presence of terpenoids in large quantities in plants, a large number of terpenoids have been extracted from marine organisms. Many terpenoids have important physiological activities and are an important source for the study of natural products and the development of new drugs (Kim et al., 2020). In addition, there were resorcinol clusters in strains B. aurantiacum N62T and B. mesophilum JCM 14063T, which were not present in the other three strains.
Carbohydrate-Active Enzymes (CAZymes)
Analysis of the genomes of genus Brumimicrobium using the CAZy database, given their polysaccharide utilization. Strain C305T, B. aurantiacum N62T, B. glaciale LMG 21434T, B. mesophilum JCM 14063T and B. salinarum KCTC 62372T contain 55, 69, 60, 61 and 48 carbohydrate-active enzymes, respectively (Fig. 4). Among these carbohydrate-active enzymes, glycosyltransferases (GTs) were the most numerous enzymes. Compared to the four reference strains, strain C305T contained 55 carbohydrate-active enzymes, including 37 GTs, which were significantly lower than B. aurantiacum N62T, B. mesophilum JCM 14063T and B. glaciale LMG 21434T. The abundance of carbohydrase in the strains contributes to their utilization of carbohydrates, which may help them survive.
Pan-genome analysis
Five strains of genus Brumimicrobium were analyzed using the BPGA pangenome. The results of the comparative analysis based on homologous proteomes exhibited 1710 core genes shared by the five species. In Supplementary Fig. S5, it can be seen that the proportion of unique genes in the core and auxiliary genes varies widely, with the core genes accounting for a higher proportion in all strains. The core genes mainly play important roles in amino acid metabolism, carbohydrate metabolism, energy metabolism, folding sorting and degradation, metabolism of cofactors and vitamins, nucleotide metabolism, replication and repair (Supplementary Fig. S6).
Chemotaxonomic characteristics
The predominant cellular fatty acids were iso-C15:0 (40.1 %),iso-C15:1 G (23.3 %) and iso-C17:0 3-OH (8.9 %),which was similar to the profile of the genus B. mesophilum JCM 14063T and B. glaciale LMG 21434T, although there were differences in the proportions of some fatty acids. Details can be found in Supplementary Table S1. Strain C305T contained phosphatidylethanolamines (PE), unidentified aminophospholipids (APL), and a kind of unidentified lipid (L2) as the major polar lipids, and three unknown lipids (L1,L3 and L4) as minor components (Supplementary Fig. S4). Strain C305T and the members of genus Brumimicrobium all contained phosphatidylethanolamine (PE), and unidentified lipid as the major polar lipids. The respiratory lipoquinone of strain C305T was MK-6, which was the same as the member of genus Brumimicrobium. The phenotypic, physiological and biochemical characteristics of strain C305T are presented in the species description, Table 1 and Supplementary Table S2. The characteristics that differentiate the new organism from its closest phylogenetic relatives B. mesophilum JCM 14063T, B. aurantiacum N62T and B. glaciale LMG 21434T are shown in Table 1 and Supplementary Table S2.
Combining phylogenetic analysis, phenotypic characteristics and chemotaxonomic studies, strain C305T should be assigned as a novel species of the genus Brumimicrobium, for which the name Brumimicrobium oceani sp. nov. is proposed.
Description of Brumimicrobium oceani sp. nov.
Brumimicrobium oceani (o.ce.a'ni. L. gen. n. oceani of the ocean).
Cells are aerobic, Gram-stain-negative, and non-gliding, rod-shaped, approximately 0.3–0.5 µm wide and 0.9–1.6 µm long. Colonies on MA were orange, opaque, circular and possess entire edges after incubation for 4 days at 28 °C. Growth occurs at 4–40 °C (optimally at 30–33 °C), pH 6.0–9.0 (optimally at pH 8.0) and with 0.5–10.0 % (w/v) NaCl (optimum 1.5–3.0 %). No growth is observed without NaCl. Positive for catalase, but negative for oxidation-fermentation test and nitrate reduction. Dextrin and gelatin are hydrolysed, while agar, starch, CM-cellulose and Tweens 20, 40, 60, 80 are not. The strain is positive for arginine dihydrolase, tryptophan deaminase, citrate utilization, gelatinase and Voges-Proskauer reaction, but negative for lysine decarboxylase, ornithine decarboxylase, H2S production, urease, indole production. Positive result in tests for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cysteine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, but negative for activities of lipase (C14), trypsin, α-chymotrypsin, a-galactosidases, b-galactosidases, b-glucuronidase, a-glucosidases, b-glucosidases, N-acetyl-b-glucosaminidase, a-mannosidase and b-fucosidase. In carbon source oxidation tests, positive for D-serine, D-sorbitol, D-mannitol, Acetoacetic acid, Acetic acid, Aztreonam Sodium. The major polar lipids are phosphatidylethanolamines (PE), unidentified aminophospholipids (APL), and a kind of unidentified lipid (L2). The respiratory quinone of C305T is MK-6. The main cellular fatty acids were iso-C15:0 ,iso-C15:1 G and iso-C17:0 3-OH.
The type strain, C305T (=KCTC 62371T =MCCC 1H00297T), was isolated from marine sediment on the coast of Weihai, China. The genomic DNA G+C content of the type strain is 35.5 mol%. The GenBank accession number for the 16S rRNA gene sequence of strain C305T is MH000430. The whole-genome shotgun projects of Brumimicrobium oceani C305T have been deposited at DDBJ/ENA/GenBank under the accession number QFRJ00000000.