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. Genome-wide information is used to construct the phylogenetic tree. Strain GXQ1321T and reference strain SST-8T clustered together to form a branch, which proved that strain GXQ1321T was the closest relative to reference strain B. samyangense SST-8T (Fig. 2). 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.
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 |
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. 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, whereas the B. samyangense SST-8T and B. rongguiense 5221T strains did not contain the ectoine gene (Table S2). 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 (Pei S et al. 2020). This result also proved that strain GXQ1321T belonged to the genus Brevibacterium.
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 |
The results of the pan-genomic comparison between strain GXQ1321T and its reference strains were OrthoVenn3 (Fig. S4). The strain GXQ1321T had a total of 2243 gene clusters, while the other two reference strains had 2298 and 1790 gene clusters, respectively. The results showed that strains GXQ1321T and B. samyangense SST-8T had the highest similarity, with a total of 594 gene clusters, which exceeded the 86 gene clusters of strains GXQ1321T and B. rongguiense 5221T. Analysis of the bacteria revealed that the three strains share a "core" genome consisting of 1,548 homologous gene clusters, most of which code for proteins with functions related to cellular metabolism and specialised membrane exchange systems. Strain GXQ1321T and strain B. samyangense SST-8T showed the deepest colour representation in the pin-to-pair orthogonal heat maps of the three genomes, indicating a close relationship between them (Fig. S5). Based on the above analysis, strain GXQ1321T and reference strain SST-8T were more closely related, which was also confirmed by the genome-wide evolutionary tree.
A total of 2,061 genes involved in six pathways were detected in the KEGG database. Of these, metabolism was the most common, including amino acid metabolism, carbohydrate metabolism, cofactor and vitamin metabolism, and energy metabolism (Fig. S6). 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.
The characteristics of nitrogen and sulphur metabolism in subsystems were compared and analysed. Strains B. samyangense SST-8T and B. rongguiense 5221T lacked genes related to cyanate hydrolysis, nitrate and nitrite antimonization, and only ammonia assimilation genes. On the contrary, strain GXQ1321T contained genes related to cyanate hydrolysis, nitrate and nitrite antimonization and ammonia assimilation. In the characteristics of sulfur metabolism, both strain GXQ1321T and the reference strains B. samyangense SST-8T and B. rongguiense 5221T contained thioredoxin-disulfide reductase, but the reference strain B. samyangense SST-8T contained genes related to organic sulfur assimilation. Therefore, B. samyangense SST-8T had the highest abundance, with a total of 11 genes related to sulfur metabolism (Table S4).
According to the analysis of the NCBI database, we found that strain GXQ1321T contained the transporters ABC(WP_349829439.1), BCCT(WP_349828541.1), MFS(WP_349829450.1), NCS2(WP_349828315.1), ACR3(WP_349826989.1), etc., which are basically the same as the transporters contained in Brevibacterium isolated from the sea, which further confirmed that strain GXQ1321T belongs to a genus of Brevibacterium. Among them, BCCT contributes to the accumulation of compatible solutes betaine, carnitine and choline. This is not only beneficial for maintaining the osmotic balance of the cell, but also acts as a stabiliser for proteins and cellular components, preventing denaturation and resisting the effects of high ionic strength. ABC transporters may be related to their ability to transport and metabolise various toxic substances and chitin, amylopectin, cellulose and starch. The Rast analysis showed that 235 subsystems existed in strain GXQ1321T, and 15 glycoside hydrolases (GH), 26 glycosyltransferases (GTs), 1 carbohydrate binding module (CBM), 14 carbohydrate esterases (CEs) and 8 auxiliary activities (AAs) were identified. Since lignocellulose is abundant in plants growing in Marine surface sediments, the coexistence of these genes suggests that they play an important role in the breakdown and modification of carbohydrates in Marine surface sediments. This was consistent with our subsequent functional assay finding that strain GXQ1321T had the ability to produce amylase and cellulase.
MFS promotes the transmembrane transport of solutes such as sugars, drug molecules, peptides, tricarboxylic acid cycle metabolites, organic anions and inorganic anions under electrochemical gradients. This corresponds to the maximum "amino acid transport and metabolism" shown in COG. At the same time, KEGG metabolic pathway analysis revealed that strain GXQ1321T contained the SecE gene. The Sec pathway is the first secretory pathway discovered in bacteria to export proteins across the plasma membrane to the periplasm and outer membrane of bacteria. The SecE gene is a gene associated with extracellular transport of cells, possibly because most marine Brevibacterium release a class of macromolecular protein heteropolysaccharide extracellular polymers into the environment to prevent ice crystal formation and adapt to low temperatures. We also identified genes related to salt tolerance and alkaline resistance in the gene annotation results of strain GXQ1321T, including the Na+/H + reverse transporters NhaC and NhaD, and the Na+-driven multidrug efflux pump, the DinF/NorM/MATE family. The results are also consistent with previous physiological experiments testing the bacteria's tolerance to saline alkaline conditions.
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. S7), 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. S8). 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. According to the appearance of the transparent circle, we found that strain GXQ1321T had the function of producing amylase, amylase and ferriferite.
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 | - | - | + |
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. S9). 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 S5). 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) and two unknown glycolipid (UG) were the major polar lipids of strain GXQ1321T (Fig. S10). 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.
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) and two unknown glycolipid (UG). 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.
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.