Genomic analysis
The strain GXQ1321T (GenBank accession number JBDIUY000000000) has a genomic length of 3347008 bp and 69.6% G + C. The genome contains 26 overlapping groups with maximum and minimum contigs lengths of 315,757 and 1251bp, N50 values of 227,695 bp, N90 values of 94,674 bp, and L50 values of 6, respectively. With maximum and minimum contig lengths of 315,757 and 1251 bp, N50 values of 227,695 bp, N90 values of 94,674 bp, and L50 values of 6, the genome is composed of 26 overlapping groups. Table 1 shows that the ANI values of B. rongguiense 5221T, B. samyangense SST-8T (96.77%), and GXQ1321T were all below the species criterion (ANI 95%). dDDH calculation showed that the DNA affinity of GXQ1321T and B. samyangense SST-8T (96.77%) was 21.2%. DNA affinity with B. rongguiense 5221T (96.32%) was 15.3%. The results were below the 70% threshold required to discriminate between species. The AAI values of GXQ1321T, B. samyangense SST-8T (96.77%) and B. rongguiense 5221T were also below the species threshold (AAI 95%) (Fig. S3). The results showed that GXQ1321T belongs tothe genus Brevibacterium. Several features of the genome of strain GXQ1321T distinguished it from other Brevibacterium strains by annotation and analysis of the genome (Table 2 and Table S1). The annotation results showed that GXQ1321T, B. samyangense SST-8T and B. rongguiense 5221T all contained terpene and NAPPA genes. The GXQ1321T strainn contained the ectoine gene, whereas the B. samyangense SST-8T and B. rongguiense 5221T strains did not contain the ectoine gene (Table S2). Annotation results from the antiSMASH 7.1 database showed that GXQ1321T contained a terpene gene cluster with 33% similarity to carotenoids. The yellow-green colour of the GXQ1321T colony was also consistent with the phenotypic characteristics. In addition, GXQ1321T also contains 75% ectoine. Ectoine helps organisms withstand high osmotic pressure environments. Ectoine protects enzymes, membranes and whole cells from the stresses of salt, heating, freezing and drying. The ability of strain GXQ1321T to endure in marine sediments was also verified by this outcome. This gene cluster is present in the genus Brevibacterium, which was isolated from a harsh environment [7]. This result also proved that strain GXQ1321T belonged to the genus Brevibacterium. We discovered that "amino acid transport and metabolism" was the largest of the known coding proteins of strain GXQ1321T based on a COG classification analysis (Table S3). Since strain GXQ1321T was isolated from marine surface sediments, we hypothesised that ectoine secreted by GXQ1321T may affect the growth and development and stress resistance of marine sediment plants.
Table 1
ANI and dDDH values between strain GXQ1321T and other closely related strains.
Strain 1 | Strain 2 | ANI (%) | dDDH (%) | 16S rRNA gene identity (%) |
GXQ1321T | B. Rongguiense 5221T | 73.91 | 15.3 | 96.32 |
B.samyangense SST-8T | 77.14 | 21.1 | 96.77 |
Table 2
Comparison of the genomic characteristics of GXQ1321T and related members of the genus Brevibacterium. +, positive; –, negative.
Genes putatively encoding | GXQ1321T | B. samyangense SST-8T | B. Rongguiense 5221T |
GenBank accession number | JAOEFD000000000 | BAAANO000000000 | JACCFQ010000000 |
Genome size (bp) | 3347008 | 3394428 | 3117581 |
DNA G + C content (mol%) | 69.6 | 68.95 | 72.4 |
N50 value (bp) | 139003 | 41933 | 42508 |
L50 value (bp) | 9 | 8 | 23 |
Number of coding sequences | 3088 | 3197 | 2904 |
features in the draft genome | 3147 | 3252 | 2952 |
Number of rRNAs | 59 | 55 | 48 |
Number of Subsystems | 235 | 238 | 236 |
Morphological, physiological, and biochemical characteristics
The GXQ1321T strain's colony was circular, convex, golden, and free of diffuse pigment. Its margins were unbroken. Following 48–72 hours of cultivation at 30 ℃, the strain's cells had a short rod-like morphology (0.51–0.59 × 1.1–1.6 µm) (Fig. S4), with a colony diameter of 1.2–2.3 mm. Strain GXQ1321T was positive for Gram staining and aerobic. Transmission electron microscopy revealed that strain GXQ1321T was flagellated and nonmotile (Fig. S5). This is similar to the reference strains, the reference strains B. samyangense SST-8T and B. rongguiense 5221T have no flagellum and are non-motile, while most strains of the genus Brevibacterium have no flagellum. The strain was grown on actinomycetes culture media for three days at 30°C. The colonies were 1.1–1.8 mm in diameter and yellow in colour. After one month of observation, strain GXQ1321T grew at 4–50℃ (optimal 30 ℃). The concentration of NaCl tolerance ranged from 0 to 20% (4% was optimal). Catalase detection results of strain GXQ1321T were negative. The pH range in which the strains can grow is 4.0–10 (7.0 is optimal). Table 3 illustrates the observed variations in physiological characteristics between GXQ1321T and the reference strains B. samyangense SST-8T and B. rongguiense 5221T. Strain GXQ1321T and the reference strains B. samyangense SST-8T, B. rongguiense 5221T were sensitive to amoxicillin, streptomycin, erythromycin, rifampicin, neomycin and chloramphenicol. Compared to the reference strain B. rongguiense 5221T, strain GXQ1321T and reference strain B. samyangense SST-8T were not resistant to neomycin and gentamicin. Table 3 displays the differences in API 20NE, API 50CH, API ZYM, and Biolog GEN III as well as the physiological and biochemical features of strain GXQ1321T compared to reference strains B. samyangense SST-8T and B. rongguiense 5221T. The peptidoglycan of strain GXQ1321T is consistent with the peptidoglycan of the reference strains B. samyangense SST-8T and B. rongguiense 5221T, whole cell hydrolysates containing meso-diaminopimelic acid (Fig. S6). Anteiso-C19:0 (27.28%), anteiso-C15:0 (18.97%), anteiso-C17:0 (15.95%), and iso-C16:0 (12.21%) were the major fatty acids (> 10%) of strain GXQ1321T. The major fatty acids (> 10%) of B. samyangense SST-8T were anteiso-C17:0 (35.3%), anteiso-C15:0 (29.9%) and iso-C15:0 (15.5%). The major fatty acids (> 10%) of B. rongguiense 5221T were anteiso-C15:0 (46.2%), anteiso-C17:0 (39.3%) and C16:0 (11.9%) (Table S4). The main fatty acids of strain GXQ1321T were different from those of B. samyangense SST-8T and B. rongguiense 5221T. Phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), three phosphoglycolipids (PGL), one unknown phospholipid (UP), and one unknown glycolipid (UG) were the major polar lipids of strain GXQ1321T (Fig. S7). Compared to GXQ1321T, the major polar lipid of B. samyangense SST-8T was phosphatidylglycerol (PG), and the major polar lipids of B. rongguiense 5221T were phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), and three types of phosphoglycolipids ( PGL). The respiratory quinones of strain GXQ1321T were mainly MK-8 (90%). The reference strains B. samyangense SST-8T and B. rongguiense 5221T breathed predominantly quinones as MK-8, consistent with most of the genus Brevibacterium. The results of physicochemical experiments demonstrated that strain GXQ1321T could be distinguished from the reference strains B. samyangense SST-8T and B. rongguiense 5221T. The growth rate of strain GXQ1321T on actinomycetes culture medium was higher than the reference strains B. samyangense SST-8T and B. rongguiense 5221T. The colony colour and moisture of strain GXQ1321T were also different from the reference strains B. samyangense SST-8T and B. rongguiense 5221T. Strain GXQ1321T and the reference strains B. samyangense SST-8T, B. rongguiense 5221T were sensitive to amoxicillin (10 µg/tablet), streptomycin (10 µg/tablet), erythromycin (15 µg/tablet), rifampicin (5 µg/tablet), neomycin (30 µg/tablet) and chloramphenicol (30 µg/tablet). Strain GXQ1321T and the reference strains B. samyangense SST-8T were resistant to neomycin (30 µg/tablet) and gentamicin (10 µg/tablet), while B. rongguiense 5221T was resistant to neomycin and gentamicin. Following an analysis of the strain's physiology, chemistry, and phylogeny, it was concluded that strain GXQ1321T represents a novel species within the Brevibacterium genus, designated as Brevibacterium litoralis sp. nov.
Table 3
Differential physiological characteristics of GXQ1321T from the closely related species B.samyangense SST-8T and B. Rongguiense 5221T. All data presented are from this study. +, positive; –, negative;W, weak reaction.
Characteristic | GXQ1321T | B.samyangense SST-8T | B. Rongguiense 5221T |
Colony color | Light-yellow | Bright-yellow | Milky - white |
Motility | - | + | - |
pH(optimum) | 4.0–10.0(7.0) | 6.0-11.5(10.0) | 5.0-9.5(7.5) |
Temperature (optimum) (°C) | 4–50(30) | 10–45(30) | 10–40(35) |
NaCl (optimum) (%, w/v) | (0–20)4.0% | 0–15(7.5) | (1–12)4% |
Assimilation of (API 20NE): | | | |
Urea | w | - | + |
Maltose | - | w | + |
Gelatin | + | - | - |
Esculin | + | - | - |
Acid production from (API 50CH): | | | |
D-Mannose | - | + | + |
D-GALactose | + | - | - |
D-MELibiose | + | - | - |
L-RHamnose | - | - | + |
D-Fruetose | - | - | + |
Arbutin | + | w | - |
Carbon source utilization (Biolog GEN III): | | | |
α-D-Glucose | + | - | - |
D-Salicin | - | - | w |
Dextrin | + | + | - |
Gentiobiose | + | - | - |
D-Turanose | + | + | - |
α-D-lactose | + | + | - |
L-Fucose | + | + | - |
Inosine | + | + | - |
D-Mannitol | + | + | - |
D-arabitol | + | + | - |
L-Aspartic acid | + | - | - |
Gelatin | + | - | - |
Pectin | + | - | - |
L-Alanine | - | + | + |
Sodium citrate | W | - | + |
Sodium formate | W | - | + |
Disodium-D,L-malate | - | - | + |
Inulin | - | + | - |
Enzymology (API ZYM): | | + | |
Acid phosphatase | - | | + |
Valine arylamidase | + | - | - |
Cysteine arylamidase | + | W | - |
Trypsin | - | W | + |
β-glucosidase | - | - | + |
Antibiotic tolerance: | | - | |
Gentamicin | - | | + |
Neomycin | - | - | + |
Description of Brevibacterium litoralis sp. nov
Brevibacterium litoralis sp. nov (li.to.ra′lis. L. masc. adj. litoralis of or belonging to the sea shore).
The strain is an aerobic, Gram-positive actinomycetes that is non-motile and does not generate spores. The cells measured 0.51–0.59 × 1.1–1.6 µm and had a short rod shape. The colony is yellow, round, convex, with intact margins and does not produce diffuse pigments. Negative for catalase. The optimal growth conditions for the organism in question are 4–50 ℃, pH 4.0–10.0, and NaCl concentration ranging between 0–20% w/v (optimum 4.0%). Maltose could not be hydrolyzed by the strain, while gelatin and aesculin could. Arbutin, D-galactose, and D-meliose were converted to acid. Dextrin, L-fructose, L-rhamnose, D-maltose, D-trehalose, myo-inositol, D-cellulobiose, sucrose, D-terabiose, stachyose, α-D-lactose,glycerol, D-glucose-6-phosphate, D-fructose-6-phosphate, β-methyl-D-glucoside, D-salicin, N-acetyl-D-glucosamine, alpha-D-glucose, N-acetyl-β-D-mannosamine, D-mannose, gelatine, D-fructose, sodium lactate, D-sorbitol, inosine, D-mannitol, D-arabitol, D-aspartate, L-alanine, gentiobiose, L-aspartic acid, L-glutamic acid, L-histidine, L-pyroglutamic acid, L-serine, pectin, D-galacturonic acid, L-galactonic acid lactone, D-glucuronic acid, D-saccharic acid, L-lactic acid, α-ketoglutaric acid, D-malic acid, D-lactic acid methyl ester, L-malic acid, Tween 40, propionic acid, acetic acid, formic acid can be used as a sources of carbon. In accordance with the API ZYM test paper, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, naphtho-AS-BI-phosphohydrolase were all positive. Lipase (C14), chymotrypsin, β-fucosidase, alpha-mannosidase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-glucosaminase were negative.
The enzymes produced by this strain were esterase, amylase and cellulase. The strain was resistant to neomycin, amoxicillin, streptomycin, chloramphenicol, rifampicin, novobiocin, gentamicin and erythromycin. The main respiratory quinone was MK-8 (90%). The main polar lipids of strain GXQ1321T were phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), three phosphoglycolipids (PGL), one unknown glycolipid (UG) and one unknown phospholipid (UP). The whole cell hydrolysates containing meso-diaminopimelic acid. Anteiso-C19:0 (27.28%), anteiso-C15:0 (18.97%), anteiso-C17:0 (15.95%), and iso-C16:0 (12.21%) were the major fatty acids (> 10%) of strain GXQ1321T. The strain in question has a DNA G + C content of 69.6 mol%. Strain GXQ1321T (= MCCC 1K08964T = KCTC 59167T) was the first Brevibacterium isolated from surface sediments in Beihai, Guangxi. The 16S rRNA gene for the strain GXQ1321T has been assigned the GenBank accession number OR661284. The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JBDIUY000000000.