Oceanimonas pelagia sp. nov., a novel biosurfactant producing and plastic-degrading potential bacterium isolated from marine coastal sediment

doi:10.21203/rs.3.rs-3608616/v1

Download PDF

Research Article

Oceanimonas pelagia sp. nov., a novel biosurfactant producing and plastic-degrading potential bacterium isolated from marine coastal sediment

https://doi.org/10.21203/rs.3.rs-3608616/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 07 Mar, 2024

Read the published version in Antonie van Leeuwenhoek →

You are reading this latest preprint version

A marine bacterial strain, designated as NTOU-MSR1^T, was isolated from the marine sediment of Taiwan's northern coast. This bacterium is Gram-stain-negative, aerobic, and motile. It possesses a single flagellum, and its cells are rod-shaped, with an approximate width of 0.5-0.6 µm and a length of 1.8-2.0 μm. Growth is observed at 10-45℃ with an optimum at 30℃. The pH range for growth is 7.0-10.0, with optimal growth at pH 7.0-8.0. The cells can grow with a concentration of NaCl up to 12%. In the cell membrane, the predominant fatty acids are C_16:1ω7c, C_18:1ω7c, and C_16:0. The overall genome relatedness indices indicate that strain NTOU-MSR1^T has an average nucleotide identity (ANI) of 87.88% and a digital DNA-DNA hybridization (dDDH) value of 35.40% with its closest related species, O. marisflavi 102-Na3ᵀ, both values failing below the 95% and 70% species threshold. This indicates that strain NTOU-MSR1^T is a novel member belonging to the Oceanimonas genus. The genomic DNA G+C content is 61.0 mol%. Genomic analysis uncovers the presence of genes associated with the catechol branch of β- ketoadipate pathway for polycyclic aromatic hydrocarbons (PAH) degradation, heavy metal resistance, production of poly(hydroxybutyrate)(PHB). In addition, strain NTOU-MSR1^T has been found to possess the ability to synthesize biosurfactants and have a potential ability of plastic degradation. A proposed name, Oceanimonas pelagia, was assigned to this novel species, and the type strain of this bacterium was designated as NTOU-MSR1^T (=BCRC 81403ᵀ=JCM 36023ᵀ).

Marine sediment

Oxford Nanopore Technologies

biosurfactants

plastic degradation

Oceanimonas, a genus associated with the phylum Pseudomonadota, was initially subject to a certain degree of taxonomic misinterpretation. The genus was erroneously classified as part of the Pseudomonas genus, subsequent studies led to the reclassification and correction in 2001, officially recognizing it as Oceanimonas (Notification that new names and new combinations have appeared in volume 51, part 1, of the IJSEM, 2001; Baumann et al., 1972; Brown et al., 2001). According to the most recent data, only four species have been characterized within this genus (https://lpsn.dsmz.de/genus/oceanimonas; LPSN), and these species have been discovered in a variety of sample types, ranging from the mud of estuaries to seawater, and sediment samples from coastal beach (Ivanova et al., 2005; Lee et al., 2018). It was first described and misclassified as Pseudomonas by Baumann et al. in 1972 (Baumann et al., 1972). In 2001, the type species [Pseudomonas] doudoroffii was reclassified and renamed as Oceanomonas. Additionally, Brown et al. discovered and reported a new phenol-degrading bacterium, Oceanomonas baumannii (Brown et al., 2001). In the same year, the genus Oceanomonas was further revised and became Oceanimonas (Notification that new names and new combinations have appeared in volume 51, part 1, of the IJSEM, 2001). Subsequently, O. smirnovii and O. marisflavi were isolated from a seawater sample near the Black Sea and a coastal beach sediment sample in Taean, Republic of Korea (Ivanova et al., 2005; Lee et al., 2018). Recently, strains belonging to the Oceanimonas genus have been discovered on the surface of plastisphere (Saini & Bhadury, 2022) and some strains have the ability to degrade low-density polyethylene (LDPE) (Joshi et al., 2022).

However, to date, genomic research on the genus Oceanimonas has been limited by the availability of only one complete genome obtained from the National Center for Biotechnology Information (NCBI), specifically Oceanimonas sp. GK1 (accession number: CP003171). In this study, a novel bacterium designated as NTOU-MSR1^T, was isolated from Taiwan's Northern Coast. The bacterium's genome was sequenced using both Oxford Nanopore Technologies and the Illumina NovaSeq 6000 platform. A hybrid genome assembly approach was employed to obtain the complete genome sequence. This achievement marks a significant contribution to the genomic understanding of this genus. Furthermore, a comprehensive investigation of the physiological, biochemical, and morphological characteristics of the NTOU-MSR1^T was conducted. Additionally, an emulsification and plastic degradation test were performed to assess the biosurfactant production and potential plastic degradation capacity of the strains and reference species within the Oceanmonas genus. The findings from this study have the potential to advance the current understanding of the Oceanimonas genus.

Sample collection and bacterial collection

A marine sediment sample was collected from a depth of 1 m from Taiwan’s Northern Coast (coordinates: 25°08'19.9" N 121°48'09.8" E). The sediment sample was serially diluted using sterile seawater, and the dilutants were spread on tryptone soya broth agar plates (TSB; Tryptone 17.0 g/L, Soya peptone 3.0 g/L, Sodium chloride 5.0 g/L, Dextrose (Glucose) 2.5 g/L, Dipotassium hydrogen phosphate 2.5 g/L, with supplementation of 3% (w/v) sodium chloride; HIMEDIA, Mumbai, India). The agar plates were then incubated at 25℃ for five days. A light pink colony of strain NTOU-MSR1^T was selected and purified on the new TSB agar. Strains were kept in 25% (v/v) glycerol and preserved at -80℃. To compare species within the Oceanimonas genus, we obtained type strains of Oceanimonas doudoroffii DSM 7028^T, Oceanimonas baumannii DSM 15594^T, and Oceanimonas marisflavi DSM 106032^T from the Leibniz Institute DSMZ (Braunschweig, Germany) through the Bioresource Collection and Research Center (BCRC; Taiwan). These strains served as experimental controls for all physio-biochemical experiments. Staphylococcus epidermidis F-1, isolated from the gut of the olive flounder (Paralichthys olivaceus), was used as a control in the plastic degradation potential test.

Genome sequencing, assembly and annotation

Genomic DNA was extracted with Puregene Yeast/Bact. Kit B (QIAGEN, Valencia, CA, USA) following the manufacturer’s protocol. For whole genome sequencing, the quantification of the genomic DNA sample was evaluated using a Qubit 4 Fluorometer (Invitrogen™, Carlsbad, CA, USA). Following quantification, the DNA was subjected to sequencing procedures using the Nanopore MinION platform and the Illumina NovaSeq 6000 platform (Taiwan Genome Industry Alliance Inc., Taipei, Taiwan). The sequencing process yields a total of nearly 9 gigabytes (Gb) and provides a 2188-fold coverage. Subsequently, a de novo assembly approach was adopted utilizing Flye v 2.9 (Kolmogorov et al., 2020), followed by one round of Medaka v 0.4 (https://github.com/nanoporetech/medaka) polishing and Homopolish v 0.34 polishing, thereby resulting in a preliminary assembly (Huang et al., 2021). To enhance the precision of the assembly further, the sequence was subjected to two rounds of correction using Pilon v 1.24, coupled with Illumina short reads. The final genome information was deposited in the NCBI. (Walker et al., 2014). The genome assembly was annotated by NCBI Prokaryotic Genome Annotation Pipeline (PGAP) to understand its general genomic features (Tatusova et al., 2016). Identification of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) was carried out by the CRISPRCasFinder web server. The presence of genomic islands were detected with the IslandViewer4 web server (Bertelli et al., 2017). Potential prophage regions are identified using the PHASTER website (Arndt et al., 2016). Secondary metabolite biosynthetic gene clusters (BGCs) were detected using antiSMASH 7.0 (Blin et al., 2023). The genome of the strain NTOU-MSR1 was visualized using the Circos v0.69 (Krzywinski et al., 2009).

Phylogenetic analysis, phylogenomic analysis, and overall genome relatedness indices

For identification of strain NTOU-MSR1^T, the PCR amplification of the 16S rRNA gene region was performed using two universal primers: 27F (5′- AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′- GGTTACCTTGTTACGACTT-3′). The resulting PCR product was then sent to GENOMICS Inc. (New Taipei City, Taiwan) for sanger sequencing using the abovementioned 27F and1492R primers, as well as an additional 907R primer (5’- CCG TCA ATT CCT TTR AGT TT-3’). The partial 16S rRNA gene sequence (with a length of 1441 bp) was aligned using BENCHLING (Sequence Alignment Tool, 2023) and then subsequently queried against the Genbank database of type materials with BLASTn (Altschul et al., 1990). However, it is worth mentioning that when using the 27F primer for the Sanger sequencing, multiple signals were detected. This outcome suggests the presence of intragenomic polymorphism within the multiple copies of the 16S rRNA sequence acquired from the complete genome of strain NTOU-MSR1^T. In order to obtain near-completed 16S rRNA sequences, the consensus 16S rRNA sequence of all copies was utilized for the phylogenetic analysis. Additionally, the 16S rRNA sequence of O. baumannii ATCC 700832ᵀ was acquired from the genome assembly (GCF_002245495.1) to complement the analysis. A phylogenetic analysis based on the 16S rRNA sequence was performed using MEGA11 (Tamura et al., 2021). To establish the evolutionary position of the new stain NTOU-MSR1^T, the 16S rRNA gene sequence was compared to relative genera of Oceanisphaera and Zobellella established members within the Aeromonadaceae family. Multiple sequences were aligned using the ClustalW algorithm, and the neighbor-joining method with Jukes-Cantor model were selected to generate the neighbor-joining phylogenetic tree (Thompson et al., 2003). The bootstrap values setup was 1000 replicates. The model of Hasegawa-Kishino-Yano was also used for recovering the maximum-likelihood phylogenetic tree. A phylogenomic tree based on the whole genome sequences rooted in proteome analysis was constructed using the genomes of strain NTOU-MSR1^T and all available type strains from the genera of Zobellella and Oceanimonas within the Aeromonadaceae family. The Type (Strain) Genome Server (https://tygs.dsmz.de) (Meier-Kolthoff & Göker, 2019) was utilized for this purpose. Genomic distances were computed through the Genome Blast Distance Phylogeny (GBDP) method employing the 'coverage' algorithm and the d5 distance formula (Meier-Kolthoff et al., 2013). Consequently, the results were imported into the iTOL v.6.8 web tool to create a visual representation (Letunic & Bork, 2021). Two overall genome relatedness indices (OGRI) were employed in the further analysis, specifically digital DNA-DNA hybridization (DDH and average nucleotide identity (ANI) values (Konstantinidis & Tiedje, 2005). These were determined by juxtaposing the genome of strain NTOU-MSR1^T to genomes of other Oceanimonas species using the online genome-genome distance calculator (GGDC) server with formula 2 and the Orthologous Average Nucleotide Identity Tool software (OAT), respectively (Lee et al., 2016; Meier-Kolthoff et al., 2021).

Phenotype characterization

The strain NTOU-MSR1^T was cultured in TSB medium (sup. 3% NaCl by w/v) and incubated at 30℃ for 24 hours. Gram-staining was performed utilizing the Modified Gram Stain kit (Modified Brown & Brenn; ScyTek Laboratories, Inc., Logan, UT, USA). Cell morphology and the presence of the flagella were investigated using the transmission electron microscope (TEM) by sending the sample to the Electron Microscopy Center of Institute of Marine Biology at National Taiwan Ocean University for negative staining and imaging. Cell motility was checked by inoculating the semisolid medium (TSB sup. 0.4% agar by w/v) with an inoculating needle and incubating at 30℃ for 48 hours. The morphology and color of the colonies were determined on TSB agar after incubation at 30℃ for five days. The temperature range for growth was tested on the TSB medium (sup. 3% NaCl) at 10, 15, 25, 28, 30, 37, 40, and 45℃. The pH range for growth was examined with the TSB medium at pH 4.0–11.0 (with an interval of 1.0 pH units) with different buffer systems (pH 4.0–6.0, 0.1 M citric acid/ 0.2 M \({\text{N}\text{a}}_{2}\text{H}{\text{P}\text{O}}_{4}\); pH 7.0–8.0, 0.2 M HCl/ 0.2 M Trizma base; pH 9.0–11.0, 0.1 M Glycine/ 0.1 M Saline/ 0.1 M NaOH) (Nigam, 2007). The pH of the medium was assessed using a pH meter (Meter Cyber Scan pH510; Eutec Instruments, Nijkerk, Netherlands) prior to the use, and subsequently, the medium was sterilized through filtration using 0.22 µm Millex-GS sterile filters (Merck Millipore Ltd., Tullagreen Townland, Co. Cork, Ireland). The NaCl tolerance was determined with modified TSB medium supplemented with 0–15% (w/v) NaCl. The temperature, pH, and NaCl tolerance tests were performed on the 96-well microplates. The growth of the isolated was monitored at an optical density of 600 nm by using the multi-detection microplate reader (BioTek, USA) every 24 hours. The capability to grow under anaerobic conditions was assessed on TSB agar at 30℃ for 48 hours with a modified GasPak EZ Anaerobe Pouch System following the manufacturer’s protocol. Catalase production was tested by adding 3% (v/v) \({\text{H}}_{2}{\text{O}}_{2}\). Oxidase production was tested using 1% (w/v) tetramethyl-p-phenylenediamine through a commercial Oxidase Reagent Dropper (BD). Enzyme activities were studied with API 20 NE strips (bioMérieux, Marcy l’Etoile, France) according to the manufacturer’s instructions. Test of Utilization of single carbon substrate was performed by using GEN III MicroPlates (Biolog Inc., Hayward, CA, USA) following the manufacturer’s instructions.

Chemotaxonomy characterization

Chemotaxonomic characteristics were examined by identifying the fatty acid of the cell membrane and the type of quinone. The fatty acid identification was entrusted to Bioresource Collection and Research Center. For this analysis, cultures were grown on TSB (sup. 3% NaCl) and incubated under aerobic conditions at 30℃ for 48 hours. The subsequent steps involved the extraction, saponification, and esterification of fatty acids present within the whole cells. Automated gas chromatography (GC) analysis was then carried out on the resulting fatty acid methyl esters (FAMEs), following the guidelines established by the Sherlock Microbial Identification System (MIDI) (Sasser, 1990). The quinone type of the strain was determined through in-silico analysis, which involved examining the genes associated with the biosynthesis pathway of respiratory quinones.

Emulsifier’s activity

Emulsifier activity was assessed following Cooper and Goldenberg's method proposed in 1987 (Cooper & Goldenberg, 1987). Colonies were inoculated into TSB medium (3% NaCl) and incubated at 30℃ for 24 to 96 hours. After daily incubation, the culture was centrifuged, and the resulting supernatant was collected for the emulsification test. Subsequently, diesel was added, followed by vortexing for two minutes, and the mixture was left undisturbed at room temperature. After 24 hours, the emulsion index (\({\text{E}}_{24}\)) was calculated as the emulsion layer height over the total mixture height, multiplied by 100. To establish positive and negative controls, a 10% SDS solution (w/v) and TSB medium (sup. 3% NaCl) were chosen, respectively.

Assessment of Plastic Degradation Potential

To assess the potential of bacteria to degrade Polyethylene (PE), the clear zone method was employed (Nademo et al., 2023). The culture medium was prepared using half-strength marine broth and 1.5% agar. Afterward, the medium was autoclaved at 121°C for 15 minutes, allowed to cool down to 60°C, and then supplemented with 5g/L of polyethylene particles (125µm, ALDRICH, Lot ##MKCH2624). The mixture was vigorously shaken to ensure even distribution of the particles within the medium and subsequently poured into sterile Petri plates. Once the medium had solidified, isolated colonies that had grown on marine broth agar were inoculated using a loop and then incubated at 30°C for a duration of 8 days. Following an 8-day incubation period, the plates were subjected to staining with a 0.1% Coomassie Brilliant Blue solution and subsequently destained to reveal a clear zone surrounding the colonies. The Coomassie Brilliant Blue solution was created by dissolving 0.1% (w/v) Coomassie Blue in a mixture of 40% (v/v) methanol and 10% (v/v) acetic acid. The destaining solution was prepared by combining 40% (v/v) methanol with 10% (v/v) acetic acid. Agar plates were submerged in the 0.1% Coomassie Blue R-250 solution for a duration of 20 minutes. Subsequently, the Coomassie Blue solution was decanted, and the plates were immersed in the destaining solution for another 20 minutes. Bacteria that exhibited a clear zone amidst the blue background were identified as plastic-potential degraders (Howard & Hilliard, 1999; Nademo et al., 2023).

Phylogenetic and phylogenomic analysis

The query results against the GenBank database revealed that the 16S rRNA sequence of strain NTOU-MSR1^T exhibits the highest similarity of 98.82% to O. marisflavi DSM 106032ᵀ (MG456888). Following closely is the similarity of 98.54% to O. doudoroffii DSM 7028ᵀ (AB021371.1). The phylogenetic tree based on 16S rRNA gene sequences showed that strain NTOU-MSR1^T belongs to the Oceanimoans clade, with O. marisflavi DSM 106032ᵀ and O. doudoroffii DSM 7028ᵀ as its closest relative (Fig. 1). The phylogenomic tree was consistent with the phylogenetic trees based on 16S rRNA, clustering strain NTOU-MSR1^T with O. marisflavi DSM 106032ᵀ and O. doudoroffii DSM 7028ᵀ (Fig. 2). The clustering results of the phylogenomic tree, constructed using whole genome sequences, closely resemble the findings of the phylogenetic analysis conducted using 16S rRNA gene sequences. The pairwise comparisons of digital DDH values between Oceanimonas pelagia NTOU-MSR1 and all Oceanimonas species are 32.80% (O. doudoroffii DSM 7028ᵀ), 25.90% (O. baumannii DSM 15594ᵀ), 26.60% (O. smirnovii ATCC BAA-899ᵀ), 35.40% (O. marisflavi DSM 106032ᵀ), 34.90% (O. sp. GK1), 32.50% (O. sp. MB9), and 33.10% (O. sp. NS1), individually, and these values are much lower than the recommended threshold of species (> 70%) of digital DDH [19]. Oceanimonas pelagia NTOU-MSR1 exhibits the highest ANI values of 87.88% with O. marisflavi DSM 106032ᵀ, then 87.80% (O. sp. GK1), 86.78 (O. sp. NS1), 86.66% (O. doudoroffii DSM 7028ᵀ), 86.50 (O. sp. MB9), 82.87% (O. smirnovii ATCC BAA-899ᵀ), 82.37% (O. baumannii DSM 15594ᵀ), respectively, which is below the 95% threshold established considering a species boundary. Based on the overall genome relatedness index (OGRI), the results indicate that this strain NTOU-MSR1^T can be distinguished from its closest relatives, establishing it as a novel species within the Oceanimonas genus.

Previous research has demonstrated that the plastisphere-associated Oceanimonas sp. isolate can degrade low-density polyethylene (Joshi et al., 2022). In 2022, a phylogenetic analysis of three Oceanimonas sp. isolates (NIOT.PDB3- NIOT.PDB5, accession number: MZ708724-MZ708726) from the plastisphere was conducted. These isolates showed the closest genetic similarity to O. marisflavi DSM 106032ᵀ (strain 102-Na3ᵀ) with similarities of 98.84 to 98.98% (Joshi et al., 2022). However, with the addition of strain NTOU-MSR1^T into the database, it became apparent that this novel species exhibited even higher similarity percentages, ranging from 99.23 to 99.87%. Consequently, it has been determined that all these isolates belong to this new species.

Genome analysis

The general features of strain NTOU-MSR1^T are summarized in Table S1, and the genome is visualized in Supplementary Figure S1. The complete genome of the strain NTOU-MSR1 is comprised of a single circular chromosome with a length of 3,592,406-bp long. The GC content is 61.0%. No plasmid, 1 type I-F CRISPR–Cas system and 5 prophage regions are detected. Thirteen genetic islands, which include 295 genes, are identified. The genome is estimated to be 100% complete with contamination of 0.09% based on the CheckM analysis. Totally 3,321 protein-coding sequences (CDS) are predicted, and 94 tRNA, 25 rRNA (5S, 16S, 23S), and 4 ncRNA are detected (Table S1).

The genome of strain NTOU-MSR1 ^T contains several genes associated with the catechol branch of β- ketoadipate pathway involved in the degradation of PAHs (locus tag PU634_15375 to _15380, PU634_15400 to _15410, PU634_04665). These genes include essential enzymes such as catechol 1,2-dioxygenase (catA), muconate/chloromuconate family cycloisomerase, muconolactone delta-isomerase (catC), alpha/beta hydrolase, 3-oxoadipate CoA-transferase, 3-oxoadipyl-CoA thiolase (pcaF), and CoA-transferase. Additionally, the presence of genes encoding benzoate 1,2-dioxygenase (PU634_15415 to _15425) in the genome of strain NTOU-MSR1^T indicates its capacity for aerobic benzoate degradation via the β-ketoadipate pathway. These findings highlight the metabolic capabilities of strain NTOU-MSR1^T and suggest its potential for environmental remediation and applications in addressing environmental contamination caused by PAHs and benzoate-containing substances.

This genome also contains a number of genes that are related to resistance to heavy metals, encoding copper resistance system multicopper oxidase (PU634_03685, PU634_06750), copper resistance protein B (PU634_03690, PU634_06755), cupredoxin family protein (PU634_03695, PU634_06760), heavy metal sensor histidine kinase (PU634_06770), and heavy metal translocating P-type ATPase (PU634_02520) involved in copper homeostasis mechanisms (Giachino & Waldron, 2020). Other heavy metals resistant genes encoding chromate efflux transporter (chrA, PU634_02540), two nickel/cobalt efflux protein RcnA (PU634_03740 and PU634_16635), Ni(II)/Co(II)-binding transcriptional repressor RcnR (rcnR; PU634_03735, PU634_16640), magnesium/cobalt transporter CorA (corA, PU634_09050), and CNNM family magnesium/cobalt transport protein CorC (corC, PU634_11385) are also being detected. Besides, several genes related to arsenic resistance or arsenate detoxification are detected (PU634_07750 to PU634_07760, PU634_07770), which encode organoarsenical efflux MFS transporter ArsJ (arsJ), ArsJ-associated glyceraldehyde-3-phosphate dehydrogenase, ACR3 family arsenite efflux transporter (arsB), and metalloregulator ArsR/SmtB family transcription factor. The presence of these genes indicates the potential of strain NTOU-MSR1 to survive in environments contaminated by heavy metals and toxic compounds.

Furthermore, the genes related to the biosynthesis pathway of PHB are detected (PU634_06235, PU634_06240, PU634_06250), including the genes encodings acetyl-CoA C-acetyltransferase, acetoacetyl-CoA reductase (phbB) and class I poly(R)-hydroxyalkanoic acid synthase (phaC). The genome of strain NTOU-MSR1^T also contains genes encoding a TIGR01841 family phasin protein (phaP, PU634_16465) and a hypothetical phasin family protein (PU634_06245), as depicted in Figure S2. Phasins, also known as granule-associated proteins, are surface proteins that stabilize the PHA storage granules in bacteria (Mezzina & Pettinari, 2016). A gene encoding polyhydroxyalkanoate synthesis repressor PhaR (phaR, PU634_16475), which represses and autoregulates the expression of genes encoding the phasin protein (PhaP) and PhaR itself, was also found. Interestingly, a peptidylprolyl isomerase-encoding gene (PU634_16470) is identified through NCBI PGAP; however, its relation to the PHB cluster, phasin genes, and regulatory genes remains unclear. The presence of these genes suggests that strain NTOU-MSR1 may be capable of synthesizing PHA.

The prediction of BGCs within the genome sequence is analyzed using antiSMASH 7.0, revealing the presence of 3 BGCs, which are responsible for the biosynthesis of ectoine, ribosomally synthesized and post-translationally modified peptides (RiPP)-like product, and non-ribosomal peptide synthetases (NRPS)-independent siderophore. The ectoine biosynthetic gene cluster (PU634_06015 to _06025) is responsible for the production of ectoine, which is a low-molecular-mass compatible solute. Ectoine acts as an osmoprotectant to counteract the increase in environmental salinity (Bursy et al., 2007). The genes detected in this cluster include diaminobutyrate acetyltransferase (ectA), diaminobutyrate-2-oxoglutarate transaminase (ectB), ectoine synthase (ectC), and a specialized aspartate kinase (PU634_06030) involved in ectoine biosynthesis. The RiPP-like cluster is detected based on the presence of the gene encoding 30S ribosomal protein S12 methylthiotransferase accessory factor YcaO (ycaO, PU634_02785). Additionally, the presence of a non-ribosomal peptide synthetase (NRPS)-independent, IucA/IucC-like (NI-) siderophore biosynthetic gene cluster was detected by antiSMASH 7.0, which shows high similarity (> 85%) to the vibrioferrin biosynthetic gene cluster from Azotobacter vinelandii CA (BGC0002527). It encodes two IucA/IucC family proteins (PU634_12740, PU634_12750), one MFS transporter (PU634_12745), one type III PLP-dependent enzyme (PU634_12755), and an additional hypothetical protein (PU634_12735). The presence of this cluster suggests the potential for siderophore production in order to acquire iron from the nutrient-poor marine environment. It is worth noting that this specific siderophore has not been previously reported in the Oceanimonas genus. However, further investigation is required to confirm the presence of this siderophore and identify its specific product.

Morphological, physiological, biochemical characteristics and chemotaxonomy

Strain NTOU-MSR1^T was Gram-stain-negative, aerobic, motile, and rod-shaped. The size of the cell was approximately 0.5–0.6 µm in width and 1.8-2.0 µm in length, as illustrated in Figure S3. A single flagellum was observed on one end of the bacterium, indicating a monotrichous arrangement. Colonies were convex, smooth, round with a light pink color, and approximately 8.0 mm wide after three days of incubation at 30°C. Growth between 10–45°C was observed on TSB agar (Optimum at 30°C). The pH range for growth was 7.0–10.0 (optimal growth at pH 7.0–8.0). The strain grew in TSB broth containing 0%-12% (w/v). Catalase and oxidase activity were positive. The reduction of nitrates to nitrites was positive. Assimilations of malic acid and trisodium citrate were observed. The strain NTOU-MSR1^T exhibited the ability to utilize D-aspartic acid, L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, L-histidine, L-serine, methyl pyruvate, L-lactic acid, citric acid, L-malic acid, bromo-succinic acid, ɑ-hydroxy-butyric acid, β-hydroxy-D, L-butyric acid, ɑ -keto-butyric acid, propionic acid, acetic acid, and formic acid as single carbon. The differential morphological and physio-biochemical characteristics of strain NTOU-MSR1^T and the experimental control strains were summarized in Table 1. Table S2 presented detailed information regarding the results of the BIOLOG GEN III Microplates analysis conducted on strain NTOU-MSR1^T and other strains. The fatty acid identification result showed that the cell membrane of the strain NTOU-MSR1^T primarily consisted of C_16:1ω7c at 34.53% ± 1.22%, C_18:1ω7c at 27.78% ± 1.38%, and C_16:0 at 22.38% ± 0.77% (Table S3). The comparison of the cellular fatty acid profiles between strain NTOU-MSR1^T and related species indicates that they share a similar composition of major fatty acids (Lee et al., 2018). Through in-silico analysis, key genes encoding the enzymes involved in the biosynthetic pathway were detected within the genome of the strain NTOU-MSR1^T (Table S4). Previous reports have indicated that ubiquinol-8 (Q-8) was the predominant quinone in O. marisflavi DSM 106032^T (strain 102-Na3^T) (Lee et al., 2018). Based on the result of in silico analysis, it suggests the isoprenoid quinone type of strain NTOU-MSR1^T might be ubiquinone.

Table 1

Difference of morphological, physio-biochemical characteristics of strain NTOU-MSR1^T and closely related *Oceanimonas* species. Strains: 1, stain NTOU-MSR1^T; 2, *Oceanimonas doudoroffii* DSM 7028^T, and; 3, *Oceanimonas baumannii* DSM 15594^T; 4, *Oceanimonas marisflavi* DSM 106032^T. +, Positive reaction; -, Negative reaction; W, Weakly positive reaction.
Characteristics	1	2	3	4
Colony Color	Pink	White	White	Pink
Temperature Range for Growth (℃)	10–45	10–45	10–45	10–45
pH Range for Growth	7–10	8–10	6–10	7–10
Oxidase	+	+	+	+
Catalase	+	+	+	+
Enzyme Activities (API 20NE):
Reduction of Nitrate	+	+	-	+
Gelatin Hydrolysis	-	-	-	+
Phenylacetate	-	+	-	-
Carbon-source utilization (BIOLOG GEN III Microplate):
D-Fructose	-	+	-	+
D-Serine	-	+	+	-
Glycyl-L-Proline	-	+	+	+
D-Glucuronic Acid	-	w	-	+
D-Lactic Acid Methyl Ester	-	+	+	+
Citric Acid	+	+	+	+
⍺-Keto-Glutaric Acid	-	+	+	-
D-Malic Acid	-	+	+	+
L-Malic Acid	+	+	+	+
𝛾-Amino-Butryric Acid	-	+	+	+
β-Hydroxy-D, L-Butyric Acid	+	+	+	+
⍺-Keto-Butyric Acid	+	-	+	w
Acetoacetic Acid	-	+	-	+
Propionic Acid	+	-	+	-
Acetic Acid	+	+	+	+
Formic Acid	+	+	+	+
DNA G + C content (mol%)	61.0	60.0	55.5	59.5

Emulsifier’s activity and plastic degradation potential

The results of the emulsification test experiment were shown in Fig. 3A. A positive result was observed of the strain NTOU-MSR1^T, with an \({\text{E}}_{24}\) value of 19.91 ± 2.23 after one day of culturing of the supernatant of the bacterial culture. This finding indicated the potential production of the biosurfactants that was secreted into the medium. However, a decline in biosurfactant production was observed during a 4-day incubation period (Fig. 3), suggesting possible instability of the biosurfactants or a decrease in the specific metabolic pathway responsible for biosurfactant generation. Further investigation is required to elucidate the chemical structure and precise characteristics of the potential biosurfactants. Our research provides preliminary evidence for the presence of biosurfactants in the novel marine species NTOU-MSR1^T, highlighting its potential as a candidate for further studies related to biosurfactant research.

For detecting LDPE degradation was carried out by the clear zone method. We found that strain NTOU-MSR1^T formed detectable clear zones, whereas Staphylococcus epidermidis F-1, isolated from marine animals, did not form any (Fig. 4A). O. baumannii DSM 15594T also exhibited a slight clear zone around the colony (Fig. 4B). The clear zone experiments indicated that strain NTOU-MSR1T and O. baumannii DSM 15594^T possess plastic degradation potential. However, Staphylococcus epidermidis F-1 showed negative results. Previous research has demonstrated that the clear zone method can be utilized to identify bacterial consortia capable of degrading low-density polyethylene (Joshi et al., 2022). Therefore, the candidate strain NTOU-MSR1T merits further research in the context of plastic degradation.

In this study, the complete genome of strain NTOU-MSR1^T was sequenced and utilized for two OGRI analyses, revealing its classification as a novel species within the genus Oceanimonas. In addition, the strain NTOU-MSR1^T was subjected to morphological, physiological, and biochemical tests, which collectively allowed for its differentiation from other Oceanimonas species. The genomic analysis of the moderately halotolerant strain NTOU-MSR1 reveals the presence of genes related to the β- ketoadipate pathway of PAH degradation, heavy metals resistance, and PHB production. Additionally, a siderophore biosynthetic gene cluster was detected. Significantly, the strain NTOU-MSR1^T exhibited not only biosurfactant production but also demonstrated potential for plastic degradation. Based on the aforementioned results, the strain NTOU-MSR1^T is considered a novel species of Oceanimonas, which is named Oceanimonas pelagia, and the type strain of this bacterium was designated as NTOU-MSR1^T (= BCRC 81403ᵀ=JCM 36023ᵀ).

Description of Oceanimonas pelagia sp. Nov.

Oceanimonas pelagia (pe.la.gia. L. f. adj. pelagia of the sea) is Gram-stain-negative, aerobic, motile, monotrichous, and rod-shaped (approximately 0.5–0.6×1.8-2.0 µm in diameter). Colonies are convex, smooth, and round with a light pink color. Oxidase- and catalase positive. Growth is observed at 10–45℃ with an optimum at 30℃. The pH range for growth is 7.0–10.0, with optimal growth at pH 7.0–8.0. Cells can grow with a concentration of NaCl up to 12%. The reduction of nitrates to nitrites is positive. Assimilations of malic acid and trisodium citrate are observed. D-aspartic acid, L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, L-histidine, L-serine, methyl pyruvate, L-lactic acid, citric acid, L-malic acid, bromo-succinic acid, ɑ-hydroxy-butyric acid, β-hydroxy-D, L- butyric acid, ɑ -keto-butyric acid, propionic acid, acetic acid, and formic acid are utilized as single carbon substrates. The membrane composition of the strain is characterized by the dominant fatty acid C_16:1ω7c, C_18:1ω7c, and C_16:0. Through the in-silico method, ubiquinone is considered as its isoprenoid quinone type. Furthermore, it is noteworthy that Oceanimonas pelagia exhibits the capacity to potentially produce biosurfactants. The type strain, NTOU-MSR1^T (= BCRC 81403^T = JCM 36023^T), was isolated from a marine sediment sample of Taiwan’s Northern Coast.

Acknowledgments

The authors thank the Electron Microscopy Center of Institute of Marine Biology at National Taiwan Ocean University for technical assistance and the Biological Resource Center (BCRC) for their technical expertise in conducting fatty acid identification for this study.

Author contributions

Yang H.T. and Ho Y.N. wrote the main manuscript content. Yang H.T. isolated and the bacterium and performed all experiments. Huang Y.H. conducted the experiments to assess the bacterial plastic degradation potential. Ho Y.N. and Yang H.T. prepare all figures, as well as the supplementary file, and reviewed the manuscript.

Funding information

This study was supported by the National Science and Technology Council of Taiwan (MOST 111-2628-M-019-001-MY3).

Bacterial deposition and Data Availability

The bacterium Oceanimonas pelagia NTOU-MSR1ᵀ has been deposited in the BCRC collection with the accession number BCRC 81403, and in the JCM collection with the accession number JCM 36023. The 16S rRNA sequence and the complete whole genome sequence of Oceanimonas pelagia NTOU-MSR1^T have been deposited in the GenBank database under accession numbers OQ450183 and CP118224. The supplementary file is available in the online edition of this article.

Conflicts of interest

The authors declare that they have no conflict of interest.

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of molecular biology, 215(3), 403–410. https://doi.org/10.1016/S0022-2836(05)80360-2
Arndt, D., Grant, J. R., Marcu, A., Sajed, T., Pon, A., Liang, Y., & Wishart, D. S. (2016). PHASTER: a better, faster version of the PHAST phage search tool. Nucleic acids research, 44(W1), W16–W21. https://doi.org/10.1093/nar/gkw387
Baumann, L., Baumann, P., Mandel, M., & Allen, R. D. (1972). Taxonomy of aerobic marine eubacteria. Journal of bacteriology, 110(1), 402–429. https://doi.org/10.1128/jb.110.1.402-429.1972
Bertelli, C., Laird, M. R., Williams, K. P., Simon Fraser University Research Computing Group, Lau, B. Y., Hoad, G., Winsor, G. L., & Brinkman, F. S. L. (2017). IslandViewer 4: expanded prediction of genomic islands for larger-scale datasets. Nucleic acids research, 45(W1), W30–W35. https://doi.org/10.1093/nar/gkx343
Blin, K., Shaw, S., Augustijn, H. E., Reitz, Z. L., Biermann, F., Alanjary, M., Fetter, A., Terlouw, B. R., Metcalf, W. W., Helfrich, E. J. N., van Wezel, G. P., Medema, M. H., & Weber, T. (2023). antiSMASH 7.0: new and improved predictions for detection, regulation, chemical structures and visualisation. Nucleic acids research, 51(W1), W46–W50. https://doi.org/10.1093/nar/gkad344
Brown, G. R., Sutcliffe, I. C., & Cummings, S. P. (2001). Reclassification of [Pseudomonas] doudoroffii (Baumann et al. 1983) into the genus Oceanomonas gen. nov. as Oceanomonas doudoroffii comb. nov., and description of a phenol-degrading bacterium from estuarine water as Oceanomonas baumannii sp. nov. International journal of systematic and evolutionary microbiology, 51(Pt 1), 67–72. https://doi.org/10.1099/00207713-51-1-67
Bursy, J., Pierik, A. J., Pica, N., & Bremer, E. (2007). Osmotically induced synthesis of the compatible solute hydroxyectoine is mediated by an evolutionarily conserved ectoine hydroxylase. The Journal of biological chemistry, 282(43), 31147–31155. https://doi.org/10.1074/jbc.M704023200
Cooper, D. G., & Goldenberg, B. G. (1987). Surface-active agents from two bacillus species. Applied and environmental microbiology, 53(2), 224–229. https://doi.org/10.1128/aem.53.2.224-229.1987
Giachino, A., & Waldron, K. J. (2020). Copper tolerance in bacteria requires the activation of multiple accessory pathways. Molecular microbiology, 114(3), 377–390. https://doi.org/10.1111/mmi.14522
Howard, G., Hilliard, N. (1999). Use of Coomassie blue-polyurethane interaction inscreening of polyurethanase proteins andpolyurethanolytic bacteria. International biodeterioration & biodegradation, 43(1-2), 23-30. https://doi.org/10.1016/S0964-8305(98)00062-6
Huang, Y. T., Liu, P. Y., & Shih, P. W. (2021). Homopolish: a method for the removal of systematic errors in nanopore sequencing by homologous polishing. Genome biology, 22(1), 95. https://doi.org/10.1186/s13059-021-02282-6
Ivanova, E. P., Onyshchenko, O. M., Christen, R., Zhukova, N. V., Lysenko, A. M., Shevchenko, L. S., Buljan, V., Hambly, B., & Kiprianova, E. A. (2005). Oceanimonas smirnovii sp. nov., a novel organism isolated from the Black Sea. Systematic and applied microbiology, 28(2), 131–136. https://doi.org/10.1016/j.syapm.2004.11.002
Joshi, G., Goswami, P., Verma, P., Prakash, G., Simon, P., Vinithkumar, N. V., & Dharani, G. (2022). Unraveling the plastic degradation potentials of the plastisphere-associated marine bacterial consortium as a key player for the low-density polyethylene degradation. Journal of hazardous materials, 425, 128005. https://doi.org/10.1016/j.jhazmat.2021.128005
Kolmogorov, M., Bickhart, D. M., Behsaz, B., Gurevich, A., Rayko, M., Shin, S. B., Kuhn, K., Yuan, J., Polevikov, E., Smith, T. P. L., & Pevzner, P. A. (2020). metaFlye: scalable long-read metagenome assembly using repeat graphs. Nature methods, 17(11), 1103–1110. https://doi.org/10.1038/s41592-020-00971-x
Konstantinidis, K. T., & Tiedje, J. M. (2005). Genomic insights that advance the species definition for prokaryotes. Proceedings of the National Academy of Sciences of the United States of America, 102(7), 2567–2572. https://doi.org/10.1073/pnas.0409727102
Krzywinski, M., Schein, J., Birol, I., Connors, J., Gascoyne, R., Horsman, D., Jones, S. J., & Marra, M. A. (2009). Circos: an information aesthetic for comparative genomics. Genome research, 19(9), 1639–1645. https://doi.org/10.1101/gr.092759.109
Lee, D. W., Lee, H., Kwon, B. O., Khim, J. S., Yim, U. H., Park, H., Park, B., Choi, I. G., Kim, B. S., & Kim, J. J. (2018). Oceanimonas marisflavi sp. nov., a polycyclic aromatic hydrocarbon-degrading marine bacterium. International journal of systematic and evolutionary microbiology, 68(9), 2990–2995. https://doi.org/10.1099/ijsem.0.002932
Lee, I., Ouk Kim, Y., Park, S. C., & Chun, J. (2016). OrthoANI: An improved algorithm and software for calculating average nucleotide identity. International journal of systematic and evolutionary microbiology, 66(2), 1100–1103. https://doi.org/10.1099/ijsem.0.000760
Letunic, I., & Bork, P. (2021). Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic acids research, 49(W1), W293–W296. https://doi.org/10.1093/nar/gkab301
Meier-Kolthoff, J. P., Auch, A. F., Klenk, H. P., & Göker, M. (2013). Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC bioinformatics, 14, 60. https://doi.org/10.1186/1471-2105-14-60
Meier-Kolthoff, J. P., Carbasse, J. S., Peinado-Olarte, R. L., & Göker, M. (2022). TYGS and LPSN: a database tandem for fast and reliable genome-based classification and nomenclature of prokaryotes. Nucleic acids research, 50(D1), D801–D807. https://doi.org/10.1093/nar/gkab902
Meier-Kolthoff, J. P., & Göker, M. (2019). TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nature communications, 10(1), 2182. https://doi.org/10.1038/s41467-019-10210-3
Mezzina, M. P., & Pettinari, M. J. (2016). Phasins, Multifaceted Polyhydroxyalkanoate Granule-Associated Proteins. Applied and environmental microbiology, 82(17), 5060–5067. https://doi.org/10.1128/AEM.01161-16
Nademo, Z.M., Shibeshi, N.T., Gemeda, M.T. (2023). Isolation and screening of low-density polyethylene (LDPE) bags degrading bacteria from Addis Ababa municipal solid waste disposal site “Koshe”. Annals of Microbiology, 73(1), 6. https://doi.org/10.1186/s13213-023-01711-0
Nigam, A. (2007). Lab manual in biochemistry, immunology and biotechnology. Tata McGraw-Hill Education.
Notification that new names and new combinations have appeared in volume 51, part 1, of the IJSEM. (2001). International journal of systematic and evolutionary microbiology, 51(Pt 2), 269. https://doi.org/10.1099/00207713-51-2-269
Saini, N., Bhadury, P. 2022. Genome analysis of a plastisphere-associated Oceanimonas sp. NSJ1 sequenced on Nanopore MinION platform. IOP SciNotes, 3(4), 044601. https://doi.org/10.1088/2633-1357/ac986e
Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids, MIDI technical note 101. Newark, DE: MIDI inc.
Tamura, K., Stecher, G., & Kumar, S. (2021). MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Molecular biology and evolution, 38(7), 3022–3027. https://doi.org/10.1093/molbev/msab120
Tatusova, T., DiCuccio, M., Badretdin, A., Chetvernin, V., Nawrocki, E. P., Zaslavsky, L., Lomsadze, A., Pruitt, K. D., Borodovsky, M., & Ostell, J. (2016). NCBI prokaryotic genome annotation pipeline. Nucleic acids research, 44(14), 6614–6624. https://doi.org/10.1093/nar/gkw569
Thompson, J. D., Gibson, T. J., & Higgins, D. G. (2002). Multiple sequence alignment using ClustalW and ClustalX. Current protocols in bioinformatics, Chapter 2, . https://doi.org/10.1002/0471250953.bi0203s00
Walker, B. J., Abeel, T., Shea, T., Priest, M., Abouelliel, A., Sakthikumar, S., Cuomo, C. A., Zeng, Q., Wortman, J., Young, S. K., & Earl, A. M. (2014). Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PloS one, 9(11), e112963. https://doi.org/10.1371/journal.pone.0112963

No competing interests reported.

SupplementaryFiguresandTables.pdf

Download PDF

Journal Publication

published 07 Mar, 2024

Read the published version in Antonie van Leeuwenhoek →

Editorial decision: Revision requested
15 Nov, 2023
Editor assigned by journal
15 Nov, 2023
Submission checks completed at journal
14 Nov, 2023
First submitted to journal
14 Nov, 2023

You are reading this latest preprint version

Oceanimonas pelagia sp. nov., a novel biosurfactant producing and plastic-degrading potential bacterium isolated from marine coastal sediment

Status:

Journal Publication

Version 1

Abstract

Figures

INTRODUCTION

Materials and methods

Sample collection and bacterial collection

Genome sequencing, assembly and annotation

Phylogenetic analysis, phylogenomic analysis, and overall genome relatedness indices

Phenotype characterization

Chemotaxonomy characterization

Emulsifier’s activity

Assessment of Plastic Degradation Potential

Results and discussion

Phylogenetic and phylogenomic analysis

Genome analysis

Morphological, physiological, biochemical characteristics and chemotaxonomy

Emulsifier’s activity and plastic degradation potential

CONCLUSION

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1