Morphological, physiological and biochemical characteristics
Cells of the strain E8T were Gram-stain-negative, rod-shaped, about 0.2-0.4 µm wide and 1.0-2.5 µm long, without flagella (Fig. S1). A wide range of growth have been observed in temperature, pH and tolerance of salt, including strain E8T and related species in genus Marinomonas. The major differences of biochemical characteristics for strain E8T and related species are summarized in Table 1. All negative traits of strain E8T are provided in Table S1. According to the result of API ZYM and API 20E, strain E8T had the activity of alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase. Cellulose, starch, alginate, Tween 40, 60, 80 were not hydrolysed and negative for nitrate reduction in strain E8T.
Strain E8T was found to be susceptible to (μg per disc) penicillin (10), streptomycin (10), tobramycin (10), gentamicin (10), ampicillin (10), ofloxacin (5), carbenicillin (100), ceftriaxone (30), norfloxacin (10), cefotaxime (30), clarithromycin (CLR) (15), polymyxin B (300), chloroamphenicol (30), intermediate to neomycin (30), rifampicin (5), kanamycin (30), erythromycin (15), and resistant to vancomycin (VA) (30), tetracycline (30), lincomycin (2).
Phylogenetic and phylogenomic analyses
A nearly full-length 16S rRNA gene sequence (1425 bp) of strain E8T obtained from PCR amplification was included in the 16S rRNA gene sequences assembled from genomic sequences which contained only one complete 16s rRNA. The BLAST research in NCBI revealed that strain E8T exhibited highest similarities with M.vulgaris A79T (98.4%) and M.pontica 46-16T (97.5%). The similarity between strain E8T and the type strain JCM 20766T of the type species Marinomonas communis was 94.9%. As the topology based on maximum-likelihood phylogenetic tree of 16s rRNA genes, strain E8T was clustered with M.vulgaris A79T at a bootstrap formed a separated branch (Fig. 1). Both neighbour-joining and maximum-parsimony algorithms were used to confirm the topology of tree (Fig. S2, Fig. S3). In addition, the phylogenomic tree based on the genomic sequences with IQ Tree showed the clade formed by strain E8T and A79T could distinguished from other members in the genus and confirming the strain E8T was a member of genus Marinomonas (Fig. 2).
General genomic features
The sequence of the draft genome of strain E8T was assembled into 79 contigs with a total length of 3,285,337 bp, a contig N50 value of 126,031 bp, a contig L50 value of 8 and a mean coverage of 150 ×. The longest contig and the shortest contig were 379,893 bp and 618 bp. The DNA G+C content was 45.1mol%, which was in the middle of most of related species. A total of 3,085 genes were predicted with 2,988 protein-coding genes and 97 encode RNAs including 8 5S rRNAs, 4 16S rRNAs, 4 23S rRNAs, 77 rRNAs and 4 ncRNAs. The genome sequence of strain E8T included 31 Longinterspersed nuclear elements (LINEs), 23 Short interspersed nuclear elements (SINEs), 4 Genomics Islands (GIs), 2 CRISPR-associated genes. Further comparative general features of 30 genome sequence were shown in Table S2.
According to the genomic functions predicted, the strain E8T has been annotated with complete glycolysis, gluconeogenesis, citrate cycle (TCA cycle) and pentose phosphate pathway for central carbohydrate metabolism and UDP-N-acetyl-D-glucosamine biosynthesis, which was same as M.vulgaris A79T and M.communis JCM 20766T. The succinate dehydrogenase, cytochrome bd ubiquinol oxidase, cytochrome o ubiquinol oxidase and F-type ATPase were predicted in strain E8T and A79T but lack of cytochrome c oxidase and cytochrome bc1 complex respiratory unit which presented in JCM 20766T genome. The ackA gene was absented in strains E8T and A79T genomes which were incapable to convert acetyl-CoA into acetate as a carbon fixation pathway completed in JCM 20766T genome. The sulfate reduction ability was predicted in strain A79T with cysNC, cysN, cysD, cysNC, cysC, cysH, cysJ and cysI genes to convert sulfate into sulfide but the cysC gene was absence in strain E8T genome which may just convert sulfite into sulfide. The strain E8T was predicted to metabolism many amino acids including serine, threonine, valine, isoleucine, leucine, lysine, ornithine, arginine, proline and tryptophan. For metabolism of cofactors and vitamins, biosynthesis pathway of Pyridoxal-P (EC 1.4.3.5), NAD (EC 6.3.5.1), pantothenate (EC 6.3.2.1), coenzyme A (EC 6.3.2.5), biotin (EC 2.8.1.6), lipoic acid (EC 2.8.1.8), molybdenum cofactor (EC 2.10.1.1), siroheme (EC 4.99.1.4), heme (EC 1.3.5.3) and cobalamin (EC 6.3.1.10) were completed in strain E8T genome sequence. The genomes of three strains contain several genes responsible for choline uptake and betaine biosynthesis, including choline dehydrogenase (betA), betaine-aldehyde dehydrogenase (betB) and glycine betaine ABC transport system permease (ProU), as osmoprotectant. All genes for the synthesis of PG and PE have been found in genome of strain E8T, which were similar to other strains in genus Marinomonas.
Chemotaxonomic characterization
The major fatty acids (>10%) were C16:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c) and summed feature 8 (C18:1ω7c and/or C18:1ω6c). The summed feature 8 with ratio of 47.8%, which was the most abundant cellular fatty acid of the stain E8T, and was also the most abundant fatty acid of the related type strains M.pontica DSM 17793Tand M.communis JCM 20766T in ratio of 35.3% and 45.7%. The strain E8Tcan be distinguished from other type strains in the compounds of C17:0 with ratio of 3.1% which composition was less than 0.5% in other type strains. The composition of fatty acid summed features 8 in strain E8T was significantly higher than that in M.pontica DSM 17793T, but similar to that in M.communis JCM 20766T. The fatty acids profiles of strain E8T and reference strains M.pontica DSM 17793Tand M.communis JCM 20766T were shown in Table S3. The polar lipids were mainly composed of phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and three unidentified lipids (L1-L3), which were similar to other type strains with PG and PE as the major polar lipids (Fig. S4). The isoprenoid quinone detected in the strain E8T was consistent with the other related type strains, which was Q-8.
Comparative genome analysis
The result of genomic distance between each genome sequences showed that strain E8T has the closest distance with M.vulgaris A79T and far distance with M.agarivorans QM202T, M.flavescens ANRC-JHZ47T and M.posidonica IVIA-Po-181T (Fig. S5). The ANI and AAI values between strain E8T and M.vulgaris A79T were 78.7% and 84.3%, which below the recommended cut-off value of 95-96% (Richter and Rossello-Mora) and below the proposed cut-off for a species boundary of 85-90% (Qin et al.), respectively. Strain E8T also shared low dDDH values with M.vulgaris A79T (22.0%), below the 70% for species boundary (Goris et al. ; Meier-Kolthoff et al.). Moreover, the pairwise comparisons of the digital DDH values were shown in Table S4 and the pairwise genome comparisons in nucleotide level and protein translated genes (ANI and AAI values) were shown in Fig. 3, which suggested that strains E8T represent a putative novel species of the genus Marinomonas.
The single copy core protein tree showed that strain E8T and M.vulgaris A79T shaped a branch with close distance with bole formed by all 30 reference strains (Fig. S6). The strains M.agarivorans QM202T and M.algicola SM1966T have a long branch length with bole which may portend a rather distant phylogenetic relationships. The phylogenetic tree constructed by single-copy orthologue proteins also conformed the tree (Fig. S6). According to result of single-copy gene families, 6,806 orthogroups with 104,049 genes in orthogroups and 3,629 unassigned genes have been clustered and 1,379 orthogroups which presented in all 30 species.
The pan-genome orthologous groups (POGs) of the 30 Marinomonasstrains indicated that strain E8T had 959 core genes, 1,661 accessory genes, 219 unique genes and 10 exclusively absent genes. The plot of core-pan showed that the bifidobacteria pan-genome in Marinomonascan be considered as “open” (Fig. S7). The pipeline generated a pan-genome phylogenetic tree without outgroup based on pan-matrix data which highlighted distinct groups and confirmed in the core-genome tree (Fig. S8, S9). According to the KEGG annotation, the strains in genus Marinomonasshowed active in amino acid metabolism, carbohydrate metabolism, membrane transport, metabolism of cofactors and vitamins and xenobiotics biodegradation and metabolism. Unique genes abundantly distributed among amino acid metabolism, carbohydrate metabolism and xenobiotics biodegradation (Fig. S10). The selected type strains (12 species) related to marine plant formed 5,297 clusters, 3,916 of which are orthologous clusters (at least contains two species) and 1,381 of which are single-copy gene clusters (Table S5). Five genomes shared 1,510 protein coding regions and genome of strain E8T encodes 2,904 proteins contained 2,604 in clusters and 261 which found as singletons (Fig. 4).
The strain E8T with M.aquimarina CECT 5080T and M.vulgaris A79T have been observed a low frequency of carbohydrate transport and metabolism (COG function category G) which may associate with their oligotrophic environment. Compare to strain A79T, the strain E8T showed a relatively high count of proteins in category J, X and D which were related to genetic information transmission and transcription (Fig 5). The proteins relevant to secondary metabolites biosynthesis were observed to present in all strains. According to the antiSMASH annotations, the abundant BGCs have been predicted in the genomic sequences of 30 strains. The genomes of the strain M.mediterranea MMB-1T, M.spartinae CECT 8886T, M.posidonica IVIA-Po-181T and M.primoryensis MPKMM3633T contained the highest bioclusters, as shown in Fig. 6. In general, the genomes of most of strains were predicted to encode for ectoine, non-ribosomal polyketide synthetases (NRPS), betalactone and some other unspecified ribosomally synthesised and post-translationally modified peptide product (RiPP) cluster (Pipp-like). The Pipp-like in strain E8T has been annotated with bacteriocin which have been annotated in Marinomonas primoryensis MPKMM3633, Marinomonas pollencensis CECT 7375 and Marinomonas spartinae CECT 8887 showing a high percentage genes similarity. The MiBiG comparison showed that the bacteriocin annotated in strain E8T had the highest similarity score with compound lanthipeptide (MiBiG accession number BGC0000554) from Streptomyces filamentosus NRRL 15998. In addition, M.spartinae CECT 8886T has been annotated with a Type I PKS (Polyketide synthase) genes cluster which may mediate biosynthesis of cylindrospermopsin. According to the BAGEL4 prediction, lasso peptide was predicted in M.polaris DSM 16579T, M.mediterranea MMB-1T and M.rhizomae IVIA-Po-145T; Microcin was predicted in both M.piezotolerans YLB-05T and M.algicola SM1966T; Butyrivibriocin, garvicin_ML, bottromycin were predicted in M.rhizomae IVIA-Po-145T, M.flavescens ANRC-JHZ47T and M.atlantica Cmf 18.22T, respectively.
Description of Marinomonas algarum sp. nov.
Marinomonas algarum (al.ga′rum. L. gen. pl. n. algarum of/from algae).
Cells are Gram-stain-negative, rod-shaped, non-flagellated, catalase- and oxidase-negative and strictly aerobic bacterium, approximately 0.2–0.4 µm wide and 1.0–2.5 µm long. The colonies are white, round and smooth after incubation at 28℃ on marine agar 2216 for 48h. Growth occurs in presence of 0.5-8% NaCl (w/v; optimum 1-3%), at temperature of 15-35 ℃ (optimum 25-30 ℃), and at pH 6.0-9.0 (optimum 6.5-8.0). The strain E8T is positive to oxidize d-mannose, d-mannitol and utilize citrate, d-ribose, d-xylose, l-xylose, d-fructose, l-sorbose, esculin ferric citrate, d-lyxose, d-tagatose, potassium 5-ketogluconate, and weakly assimilated arabinose, d-turanose. The DNA G+C content is 42.8 mol%. The predominant quinone is Q-8 and the major fatty acids are C16:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c) and summed feature 8 (C18:1ω7c and/or C18:1ω6c). The main polar lipids are phosphatidylglycerol and phosphatidylethanolamine. The type strain is E8T(=KCTC 92201T =MCCC 1K07070T), isolated from red algae Gelidium amansii.
The GenBank accession number for the 16S rRNA sequence is OK444097, and the draft genome sequence of strain E8T has been deposited at GenBank under accession number JAJATW000000000.