Next Article in Journal
Fecal Microbiota Characteristics in Constipation-Predominant and Mixed-Type Irritable Bowel Syndrome
Previous Article in Journal
A Scoring System to Predict Severe Acute Lower Respiratory Infection in Children Caused by Respiratory Syncytial Virus
Previous Article in Special Issue
Prevalence and Preferred Niche of Small Eukaryotes with Mixotrophic Potentials in the Global Ocean
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Microorganisms
Volume 12
Issue 7
10.3390/microorganisms12071413
microorganisms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Description and Whole-Genome Sequencing of Mariniflexile litorale sp. nov., Isolated from the Shallow Sediments of the Sea of Japan

by
Lyudmila Romanenko
1,*,
Evgeniya Bystritskaya
1,†,
Yuliya Savicheva
1,†,
Viacheslav Eremeev
1,
Nadezhda Otstavnykh
1,
Valeriya Kurilenko
1,
Peter Velansky
2 and
Marina Isaeva
1,*
1
G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Prospect 100 Let Vladivostoku, 159, Vladivostok 690022, Russia
2
A.V. Zhirmunsky National Scientific Center of Marine Biology, Far Eastern Branch, Russian Academy of Sciences, Palchevskogo Street 17, Vladivostok 690041, Russia
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Microorganisms 2024, 12(7), 1413; https://doi.org/10.3390/microorganisms12071413
Submission received: 7 June 2024 / Revised: 9 July 2024 / Accepted: 10 July 2024 / Published: 12 July 2024
(This article belongs to the Special Issue Marine Microorganisms and Ecology)
Browse Figures
Review Reports Versions Notes

Abstract

:
A Gram-negative, aerobic, rod-shaped, non-motile, yellow-pigmented bacterium, KMM 9835T, was isolated from the sediment sample obtained from the Amur Bay of the Sea of Japan seashore, Russia. Phylogenetic analyses based on the 16S rRNA gene and whole genome sequences positioned the novel strain KMM 9835T in the genus Mariniflexile as a separate line sharing the highest 16S rRNA gene sequence similarities of 96.6% and 96.2% with Mariniflexile soesokkakense RSSK-9T and Mariniflexile fucanivorans SW5T, respectively, and similarity values of <96% to other recognized Mariniflexile species. The average nucleotide identity and digital DNA–DNA hybridization values between strain KMM 9835T and M. soesokkakense KCTC 32427T, Mariniflexile gromovii KCTC 12570T, M. fucanivorans DSM 18792T, and M. maritimum M5A1MT were 83.0%, 82.5%, 83.4%, and 78.3% and 30.7%, 29.6%, 29.5%, and 24.4%, respectively. The genomic DNA GC content of strain KMM 9835T was 32.5 mol%. The dominant menaquinone was MK-6, and the major fatty acids were iso-C15:0, iso-C15:1ω10c, and C15:0. The polar lipids of strain KMM 9835T consisted of phosphatidylethanolamine, two unidentified aminolipids, an unidentified phospholipid, and six unidentified lipids. A pan-genome analysis showed that the KMM 9835T genome encoded 753 singletons. The annotated singletons were more often related to transport protein systems (SusC), transcriptional regulators (AraC, LytTR, LacI), and enzymes (glycosylases). The KMM 9835T genome was highly enriched in CAZyme-encoding genes, the proportion of which reached 7.3%. Moreover, the KMM 9835T genome was characterized by a high abundance of CAZyme gene families (GH43, GH28, PL1, PL10, CE8, and CE12), indicating its potential to catabolize pectin. This may represent part of an adaptation strategy facilitating microbial consumption of plant polymeric substrates in aquatic environments near shorelines and freshwater sources. Based on the combination of phylogenetic and phenotypic characterization, the marine sediment strain KMM 9835T (=KCTC 92792T) represents a novel species of the genus Mariniflexile, for which the name Mariniflexile litorale sp. nov. is proposed.

1. Introduction

The genus Mariniflexile, with the type species Mariniflexile gromovii, was proposed by Nedashkovskaya et al. [1] and subsequently emended by Jung et al. [2], Jung, Yoon [3], and Park et al. [4]. The genus Mariniflexile belongs to the family Flavobacteriaceae [5], phylum Bacteroidota [6], and currently contains seven species with validly published names as listed at https://lpsn.dsmz.de/genus/mariniflexile (accessed on 3 May 2024) [7]. Bacteria of the genus Mariniflexile have been isolated from diverse marine sources, including sea urchin Strongylocentrotus intermedius [1], seawater [2,8], seawater at its confluence with a freshwater source [3,4], and an oyster [9]. It has been reported that Mariniflexile fucanivorans, a species recovered from mud formed in the process of recycling the effluent of an alginate-extraction plant, was able to degrade sulfated fucans of brown algae [10]. A unique feature of Bacteroidota is the presence of the type IX secretion system (T9SS), responsible for protein secretion through the outer membrane and involved in gliding motility, S-layer biogenesis, and biopolymer degradation [11]. In addition, the second adaptive system utilized uniquely by Bacteroidota is polysaccharide utilization loci (PULs), which allow bacteria to degrade polysaccharides in different environments. PULs are typically organized into contiguous loci where SusC/D transporter genes are adjacent to genes encoding transcriptional regulators and carbohydrate-active enzymes (CAZymes) [12].
In the present study, a Gram-negative, aerobic, non-motile bacterium, KMM 9835T, was isolated from the shallow sediment sample obtained from the Amur Bay of the Sea of Japan, Russia, and characterized. To obtain insights into the phylogenetic relationships and metabolic potential of the strain KMM 9835T among Mariniflexile species, phylogenomic and pan-genomic analyses were performed. In addition, the genomic sequence of Mariniflexile soesokkakense KCTC 32427T as a closely related phylogenetic neighbor was determined. Based on combined phylogenomic analyses and phenotypic properties, a novel species, Mariniflexile litorale sp. nov., is described.

2. Materials and Methods

2.1. Bacterial Strains

Strain KMM 9835T was isolated from the shallow sediments sampled from the Amur Bay of the Sea of Japan, Russia (42°59′23.4″ N 131°29′07.8″ E) in July 2010 by a standard dilution plating method and incubated on marine agar 2216 (MA; BD DifcoTM, Sparks, MD, USA) at 28 °C. This strain was grown aerobically on MA 2216 or in marine broth (MB) 2216 (BD DifcoTM, Sparks, MD, USA) at 28 °C and stored at −70 °C in MB 2216 supplemented with 20% (v/v) glycerol. The strain KMM 9835T has been deposited in the Collection of Marine Microorganisms (KMM), G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Vladivostok, Russia, and in the Korean Collection for Type Cultures (KCTC), Korea, as KCTC 92792T. The type strains M. soesokkakense KCTC 32427T and Mariniflexile maritimum KCTC 72895T were purchased from the Korean Collection for Type Cultures, Korea, to be used in the comparative phenotypic tests.

2.2. Phenotypic Characterization

Oxidase and catalase reactions, Gram staining, and motility (the hanging drop method) were assessed following the previously described method [13]. Gliding motility was examined as described by Bowman [14]. The morphology of cells negatively stained with 1% phosphotungstic acid was observed on carbon-coated 200-mesh copper grids using the electronic transmission microscope Libra 120 FE (Carl Zeiss, Oberkochen, Germany), provided by the A.V. Zhirmunsky National Scientific Center of Marine Biology, Far Eastern Branch, Russian Academy of Sciences. The following physiological tests, including hydrolysis of gelatin, starch, casein, Tweens 20, 40, 80, DNA, chitin, L-tyrosine, and growth at different salinities (0–12% NaCl), temperatures (4–40 °C), and pH values (4.0–11.5), were studied on the artificial seawater (ASW)-based media as described earlier [15,16]. Biochemical characteristics and enzyme activities were measured by the API 20E, API 20NE, and API ZYM test strips (bioMérieux, Marcy-l’Étoile, France) according to the manufacturer’s instructions.

2.3. Chemotaxonomic Analyses

For the lipid analyses of bacterial strain KMM 9835T, M. soesokkakense KCTC 32427T and M. maritimum KCTC 72895T were cultivated on MA 2216 at 28 °C. Lipids were extracted according to the method of Folch et al. [17]. Two-dimensional thin layer chromatography of polar lipids was performed on Silica gel 60 F254 (10 × 10 cm, Merck, Darmstadt, Germany), applying chloroform–methanol–water (65:25:4, v/v) for the first direction, chloroform–methanol–acetic acid–water (80:12:15:4, v/v) for the second one [18], and spraying with specific reagents [19]. Fatty acid methyl esters (FAMEs) were prepared following the procedure of the Microbial Identification System (MIDI) [20]. A chromatograph (Shimadzu, Kyoto, Japan) with a flame ionization detector equipped with a SPB-5 capillary column (30 m × 0.25 mm × 0.25 mkm) was used. Identification of FAMEs was carried out by comparing the equivalent chain length values and retention times of the samples to those of the standards (Standard bacterial acid methyl ester mix 47080-U, Supelco, Bellefonte, PA, USA). In addition, FAMEs were investigated using a GC-MS Shimadzu QP2020 (Shimadzu, Kyoto, Japan) with a column Shimadzu SH–Rtx–5MS (30 m × 0.25 mm × 0.25 mkm) and a temperature program from 160 °C to 320 °C at a rate of 2 °C/min). To determine the position of double bonds and methyl groups, fatty acids were analyzed as 4,4-dimethyloxazoline derivatives [21] using GC–MS with the SH–Rtx–5MS column at a temperature from 180 °C to 320 °C, 2 °C/min. Menaquinones were examined by HPLC, as described by Hirashi et al. (1996) [22]. A Shimadzu LC–30 chromatograph with a photodiode array detector (SPD–M30A) was used. Absorption spectra of lipid extracts redissolved in methanol at a concentration of 0.2 mg/mL were obtained on a Shimadzu UV–2600 spectrophotometer. The presence of flexirubin pigments was determined as described by Fautz and Reichenbach [23].

2.4. 16S rRNA Gene Sequence and Phylogenetic Analysis

Genomic DNA of strain KMM 9835T was extracted using the NucleoSpin Tissue kit (Macherey–Nagel, Düren, Germany) following the manufacturer’s instructions. The 16S rRNA gene was PCR-amplified using 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGTTACCTTGTTACGACTT-3′) primers with 5 min of denaturation (96 °C) followed by 25 cycles of 30 s of denaturation (95 °C), 30 s of annealing (55 °C), and 1 min 20 s of elongation (72 °C), finalizing with 5 min of elongation (72 °C). The obtained amplicons of standard bacterial acid were sequenced and compared with those of their closest relatives using the EzBioCloud service [24]. Phylogenies were performed on the GGDC web server (http://ggdc.dsmz.de/, accessed on 16 May 2024) [25] using the DSMZ pipeline [26] applied to a single gene. Maximum likelihood (ML) and maximum parsimony (MP) trees were inferred from the alignment with RAxML [27] and TNT [28], respectively. The neighbor-joining (NJ) tree was reconstructed with MEGA version 11 [29] using the Kimura two-parameter model of nucleotide substitutions. The robustness of phylogenetic trees was estimated by the bootstrap analysis of 1000 replicates.

2.5. Whole-Genome Sequencing, Phylogenomic, and Comparative Analyses

Genomic DNAs were extracted from strains KMM 9835T and M. soesokkakense KCTC 32427T by the NucleoSpin Tissue kit (Macherey–Nagel, Düren, Germany). The DNA quality was estimated by agarose gel electrophoresis, and the DNA quantity was measured on the Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Singapore). The DNA libraries were prepared with Nextera DNA Flex kits (Illumina, San Diego, CA, USA) and were sequenced on an Illumina MiSeq instrument using paired-end runs with a 250-bp read length. The nanopore library was prepared for KMM 9835T using EXP-NBD104 and SQK-LSK109 kits (Oxford Nanopore Technologies, Oxford, UK), according to the Native barcoding genomic DNA protocol. The reads were trimmed using Trimmomatic version 0.39 [30] and their quality assessed using FastQC version 0.11.8 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 21 August 2021 and 30 November 2023). Filtered reads of the M. soesokkakense RSSK-9T genome were assembled into contigs with SPAdes version 3.15.3 [31], and genome metrics were calculated with QUAST version 5.0.2 [32]. Dorado version 0.4.3 (Oxford Nanopore Technologies, Oxford, UK) was used with default parameters to quality filter Nanopore reads and filter out sequences 1000 bp in length. Hybrid assembly of strain KMM 9835T was performed using Unicycler v0.4.8 [33] with default parameters. Sequencing depth was estimated using samtools version 1.3 [34]. The genome completeness and contamination of strains KMM 9835T and RSSK-9T were estimated by CheckM version 1.1.3 based on the taxonomic-specific workflow (lineage Flavobacteriales) [35].
Comparisons of the Average Nucleotide Identity (ANI), Average Amino Acid Identity (AAI), and digital DNA–DNA hybridization (dDDH) values of the strains KMM 9835T and KCTC 32427T with their closest neighbors were performed with the online servers ANI/AAI–Matrix [36] and TYGS platform [37], respectively. The phylogenomic analysis was performed using PhyloPhlAn software version 3.0.1 based on a set of 400 conserved bacterial protein sequences using the RAxML program under the PROTCATLG model with bootstrapping of 100 replicates (flag −b +−100) [38].
Genome annotation was carried out using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [39], Rapid Annotation using Subsystem Technology (RAST) [40], and Prokka [41]. The circular genome of KMM 9835T was visualized using the Proksee platform [42]. Putative Horizontal Gene Transfer (HGT) events were detected via Alien Hunter [43]. CRISPR arrays and associated Cas proteins were found using CRISPR/Cas Finder [44]. Replication origin and terminus were predicted by Ori-Finder 2022 [45].
Identification of the Type 9 Secretion System (T9SS) components was conducted with MacSyFinder (TXSScan T9SS model) [46,47]. Conserved C-terminal domains (CTDs) of T9SS were searched using HMMER 3.4 (http://hmmer.org, E value of 1 × 10−2), and the hmm files of Type A (TIGR04183), Type B (TIGR04131), and ChiA (NF033708) were obtained from NCBI (accessed on 11 April 2024). CAZymes were predicted using the dbCAN3 meta server with default settings (https://bcb.unl.edu/dbCAN2/index.php, accessed on 9 December 2023) [48]. Genes predicted by no less than two algorithms integrated into the server were defined as CAZymes and selected for further analysis. CAZyme-containing gene clusters (CGCs) and PULs were annotated via the dbCAN-PUL meta server [49]. The relative abundances of CAZymes were visualized by heat maps using the pheatmap version 1.0.12 package in RStudio version 2022.02.0 + 443 with R version 4.1.3.
Metabolism estimation and pan-genomic analysis were conducted using anvi’o version 8 [50]. The FASTA files reformatted into contigs-fasta using the ‘anvi-script-reformat-fasta’ command were imported into anvi’o as contigs-db with the ‘anvi-gen-contigs-database’ command. These contigs-db’s were annotated via the ‘anvi-run-kegg-kofams’ command using the snapshot of the KEGG database accessed on 22 September 2023 [51]. An ‘anvi-estimate-metabolism’ command was consequently run with the ‘--include-metadata’ and ‘--matrix-format’ flags. The obtained data were analyzed manually using Microsoft Excel and compared with KAAS annotation data [52]. The pan-genome was reconstructed using the anvi’o workflow described at https://merenlab.org/2016/11/08/pangenomics-v2/ accessed on 28 March 2024. Cumulative curves were drawn using PanGP version 1.0.1 [53]. Pan-genome openness was estimated under Heap’s law model [54]. Fonts and sizes in all figures were edited manually in Adobe Photoshop CC 2018 for better visualization.

3. Results

3.1. Phylogenetic and Phylogenomic Analysis

Based on 16S rRNA gene sequence similarities calculated using the EzBioCloud service [24], strain KMM 9835T (OQ300347, 1398 bp) was close to M. soesokkakense RSSK-9T (96.6%), Mariniflexile sp. TRM1-10 (96.6%), M. fucanivorans SW5T (96.2%), Gaetbulibacter lutimaris D1-y4T (96.0%), and Siansivirga zeaxanthinifaciens CC-SAMT-1T (96.0%). The other representatives of Flavobacteriaceae shared values less than 96.0%, including Mariniflexile type strains: M. aquimaris HWR-17T (95.7%), M. gromovii KMM 6038T (95.7%), M. jejuense SKK2-3T (95.6%), and M. ostreae TYO-10T (94.9%). M. maritimum M5A1MT was absent from the list of the first 50 hits from the EzBioCloud 16S database.
On the 16S rRNA phylogenetic trees, the position of strain KMM 9835T was uncertain due to low bootstrap support when M. maritimum M5A1MT was included in the analysis (Figure 1). M5A1MT exclusion and TRM1-10 addition resulted in KMM 9835T clustering with Mariniflexile spp. strains (except for TYO-10T) under strong bootstrap support (Figure S1).
There are currently seven species of the genus Mariniflexile with validly published names, but only three genomes of type strains M. gromovii, M. fucanivorans, and M. maritimum are available. In this study, the genome sequence of the fourth type strain, M. soesokkakense KCTC 32427T, was obtained. The genomes of three type strains (DSM 18792T, KCTC 12570T, and M5A1MT) were retrieved from NCBI (Table 1). The phylogenomic tree based on concatenated sequences extracted from the genomes of Mariniflexile species and related taxa showed that strain KMM 9835T formed a distinct line within the genus Mariniflexile (Figure 2).
The ANI/AAI values between the genomes of strain 9835T and the type strains of M. soesokkakense KCTC 32427T, M. gromovii KCTC 12570T, M. fucanivorans DSM 18792T, and M. maritimum M5A1MT were 83.0%/83.5%, 82.5%/81.7%, 83.4%/80.7%, and 78.3%/77.6%, respectively, which were lower than the 95−96% threshold value accepted for species delineation [55]. The dDDH values (formula d4) between strain KMM 9957T and the four relatives, ranging from 24.4% (M. maritimum M5A1MT) to 30.7% (M. soesokkakense KCTC 32427T), were below the 70% threshold value accepted for species delineation [56,57]. These overall genomic relatedness indices (OGRIs) and phylogenomic position suggest that KMM 9835T represents a novel species in the genus Mariniflexile.

3.2. Genomic Characteristics and Pan-Genome Analysis of the Mariniflexile Genus

The complete genome of strain KMM 9835T was de novo assembled into one chromosome with an estimated size of 4,521,428 bp and an overall G+C content of 62.1%. The two genome-extracted 16S rRNA gene sequences were 100% identical to the PCR-amplified one (OQ300347). The genome contains 3752 protein coding sequences, 36 tRNAs, and 6 rRNA genes (two 16S-23S-5S operons). The indices recommended to evaluate the quality of the genomic data [58,59] are shown in Table 1. The observed characteristics satisfy the proposed minimal standards for the taxonomy of prokaryotes and indicate high genome quality. In total, seven Mariniflexile strains were taken for comparative genome analysis, two of which have been sequenced in this study (KMM 9835T and KCTC 32427T). In addition to the genomes of three type strains (DSM 18792T, KCTC 12570T, and M5A1MT), two high-quality genomes of Mariniflexile sp. strains (AS56 and TRM1-10) were retrieved from NCBI. Their basic genome indices are listed in Table 1. The ML phylogenomic tree, including two last genomes, clearly showed that strains AS56 and TRM1-10 might present two novel species of the genus Mariniflexile (Figure S2). The genome sequences contain from 3157 (M. maritimum M5A1MT) to 4104 (M. fucanivorans DSM 18792T) genes, from 36 (KMM 9835T and M. soesokkakense KCTC 32427T) to 41 (TRM1-10) tRNAs, and from one up to two rrn operon copies (KMM 9835T and TRM1-10).
The KMM 9835T chromosome map was built and visualized using Proksee [42] (Figure 3). Genome annotations were carried out using the RAST tool kit [40] and Prokka [41]. The first gene (dnaA) in the genome sequence was automatically assigned as the origin of replication; however, its position did not align with a GC skew plot. To identify the origin (oriC) and terminus (ter) of replication, the Ori-Finder 2022 server [45] was utilized. Regions adjacent to the mnmG and leuB genes were predicted as oriC and ter, respectively. These regions aligned with the GC skew plot and had the highest Ori-Finder scores. The chromosomal level of genomic assemblies obtained for strains KMM 9835T and TRM1-10 made it possible to estimate the exact numbers of rrn operons (Table 1); both rrn operons of KMM 9835T are located on the leading strands (Figure 3). The 13 retron-type RNA-directed DNA polymerase (EC 2.7.7.49) genes and one CRISPR-Cas region, among them seven full-length, were found in the genome strain KMM 9835T (Figure 3). Three loci had truncated ORFs.
To determine genus-related features, a pan-genome analysis of Mariniflexile species (Table 1) was performed using orthologous clustering and metabolic pathway reconstruction with the anvi`o platform [50]. The Mariniflexile pan-genome (Figure 4) comprised a total of 9163 gene clusters (distance: Euclidean; linkage: Ward) with 25,933 gene calls. The core genome included 2202 core gene clusters covering 15,113 genes, of which 1625 were single-copy genes (SCGs). The accessory shell and cloud clusters were composed of 913 (3317 genes) and 1074 (2320 genes) clusters, respectively. A unique part of the pan-genome included 4974 gene clusters (5183 genes) of singletons. The largest and smallest numbers of singletons were observed in the genomes of Mariniflexile sp. TRM1-10 (1083 clusters) and M. soesokkakense KCTC 32427T (377 clusters). The annotated singletons in the KMM 9835T genome were more prevalent and often related to transport protein systems (SusC), transcriptional regulators (AraC, LytTR, and LacI), and enzymes (Glycosylases). According to the genome size modeling, the Mariniflexile pan-genome is open with a γ value of 0.58 (Figure S3).
The completeness of metabolic pathways for all Mariniflexile genomes was calculated using the anvi’o platform [50,51] and analyzed manually using Microsoft Excel and KAAS annotation data [52]. From these results, the pathways for glycan (lipopolysaccharide), lipid, and nucleotide metabolisms were complete in all Mariniflexile strains. Their genomes encode various pathways of central carbohydrate metabolism, including the Embden-Meyerhof pathway (except for a key gene encoding hexokinase/glucokinase (EC 2.7.1.1, EC 2.7.1.2)), gluconeogenesis, the tricarboxylic acid cycle, the non-oxidative pentose phosphate pathway, 5-phospho-α-D-ribose-1-diphosphate (PRPP) biosynthesis, pyruvate oxidation, and sulfur metabolism. Moreover, the novel strains KMM 9835T and M. soesokkakense KCTC 32427T were found to contain genes encoding for the archaeal pentose phosphate pathway (M00580). The Mariniflexile genomes have the full pathways for the biosynthesis of amino acids (Pro, Lys, Thr, Ile, Ser, Trp, Leu, and His). In all genomes, the synthesis of ornithine lacks a gene for amino-acid N-acetyltransferase (EC 2.3.1.1), which is responsible for glutamate to N-acetylglutamate conversion. However, ornithine could be synthesized through the proline metabolism pathway (M00972). In addition, a gene encoding acetylornithine deacetylase (EC 3.5.1.16), which catalyzes N-acetylornithine to L-ornithine, was absent in the M. soesokkakense KCTC 32427T genome. The KMM 9835T genome contains complete degradation pathways, converting methionine to homocysteine, leucine to acetoacetate and acetyl-CoA, proline to glutamate, and a glycine cleavage system. In the pathway of tryptophan metabolism, aminomuconate-semialdehyde dehydrogenase (EC 1.2.1.32) was absent. The hydroxyproline degradation pathway lacked D-hydroxyproline dehydrogenase (EC 1.5.99.-). The pathway for histidine degradation to glutamate was not detected except for formiminoglutamase (EC 3.5.3.8). The pathways for the following cofactor and vitamin synthesis were predicted in KMM 9835T: NAD (aspartate and tryptophan-derived), coenzyme A, heme, riboflavin, tetrahydrofolate, and lipoic acid. The menaquinone biosynthetic pathway was short of a succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase (EC 4.2.99.20). The pantothenate biosynthesis pathway was incomplete due to the absence of a 2-dehydropantoate 2-reductase (EC 1.1.1.169). Siroheme is not produced since there is no sirohydrochlorin ferrochelatase (EC 4.99.1.4). The molibdenum cofactor biosynthesis pathway lacks molybdopterin adenylyltransferase (EC 2.7.7.75). Pathways for biotin synthesis were absent in KMM 9835T.

3.3. CAZymes Repertoires and Predicted PULs Analysis

The dbCAN annotation analysis revealed that strains within the Mariniflexile genus possess a highly diverse repertoire of CAZymes and PULs, which may allow them to utilize a wide range of polysaccharides. The proportion of CAZyme-encoding genes in their genomes varied from 5.3% to 7.9%, with the maximum amount predicted in strains TRM1-10 and KMM 9835T (Figure 5a). In the KMM 9835T genome, CAZyme-encoding genes reached 7.3%, comprising 164 glycoside hydrolases (GHs) classified into 52 families, 65 glycosyltransferases (GTs) into 10 families, 29 polysaccharide lyases (PLs) into 9 families, 19 carbohydrate esterases (CEs) into 9 families, and 2 auxiliary activities (AAs) into 2 families (Figure 5a).
The highest number of GHs found in KMM 9835T was related to the GH43 family (19 predicted encoding genes) containing arabinases and xylosidases (Figure 5b). That indicates the potential of the novel strain KMM 9835T to cleave arabinose moieties from xylans and pectins [60]. The greatest number of GH43-encoding genes among those of Mariniflexile spp. were also predicted in Mariniflexile sp. TRM1-10, isolated from the rhizosphere of tomato (Figure 5b). It can be assumed that strain TRM1-10 is able to utilize pectin, which is a polysaccharide primarily characteristic of land plants. The other abundant GHs of strain KMM 9835T were found to belong to GH2 beta-galactosydases (16 genes) catalyzing the degradation of different oligosaccharides. Members of the GH28 and GH92 families were also widely distributed within the KMM 9835T genome. The GH28 family was represented by 11 putative polygalacturonases that may cleave the backbone glycosidic linkages of pectin using a hydrolytic reaction [61]. The GH92 family contains α-mannosidases responsible for N-glycan cleavage that are common in many flavobacterial species [62]. All of these GHs were found within PULs (Figure 3), with the majority of GH92 members concentrated in PUL3, which is predicted to hydrolyze mucin-rich substrates. Most annotated polysaccharide lyases were classified as PL1 and PL10 families, possessing pectin and pectate lytic activities. Among carbohydrate esterases, the CE8 and CE12 families recognized for facilitating the degradation of pectin by deacetylation and demethylation [63] were the most abundant. The Mariniflexile sp. TRM1-10 and M. soesokkakense KCTC 32427T genomes shared similarities with the KMM 9835T distribution of CAZyme gene families (GH43, GH28, PL1, PL10, CE8, and CE12), which are responsible for pectin degradation. This may represent part of their adaptation strategy for facilitating microbial consumption of plant polymeric substrates in aquatic environments near shorelines and freshwater sources.
The second most frequent enzyme family in the KMM 9835T CAZome is GTs (65 encoding genes) (Figure 5a). GT2 and GT4 accounted for the highest proportion in the GT families, followed by GT51 in KMM 9835T and the related strain genomes (Figure 5b). GT2 and GT4 have been shown to perform the synthesis of α- and β-glycans and glycoconjugates [64], while GT51 enzymes, known as peptidoglycan glycosyltransferase, take part in the synthesis of murein in both Gram-positive and Gram-negative bacteria [65].
Signal peptide prediction in the KMM 9835T CAZome provided by dbCAN [48] revealed that about half of the total number of GHs, PLs, and CEs genes have signal peptide sequences targeting their products to the periplasmic space. In addition, some secretory CAZymes were predicted to contain CTDs and can be transported by the unique Bacteroidota T9SS [11,66], supporting their extracellular role in polysaccharide metabolism.
Based on biochemical characteristics (Table 2), Mariniflexile spp., except for M. gromovii KMM 6038T, showed the ability to hydrolyze starch [1,4,8,10]. Deep genomic analysis revealed that the genomes of novel bacteria KMM 9835T, M. soesokkakense KCTC 32427T, M. maritimum KCTC 72895T, and M. fucanivorans DSM 18792T contain genes encoding for the starch utilization system (Sus) [67], which explains the difference in the substrate utilization abilities of all the above-mentioned bacteria. However, these genes were not found in the M. gromovii KMM 6038T genome, which explains the difference in their substrate utilization abilities. In the KMM 9835T genome, the sus locus includes six genes, encoding outer membrane SusCD (E/F)G and periplasmic SusAB proteins. SusG is an α-amylase with CBM48, GH13_10, and C-terminal T9SS domains, which allows for hydrolysis of α-1-4 glucosidic linkages at the cell surface. Interestingly, SusC, SusD, hybrid SusE/F, and two neighboring hypothetical proteins shared 75–90% similarity with those from Tenacibaculum adriaticum DSM 18961T, Gelidibacter salicanalis PAMC21136T, and Algibacter agarivorans JCM 18285T. Moreover, this region in the KMM 9835T genome was detected as a HGT region by the AlienHunter tool in Proksee [42]. Neopullulanase SusA (GH13_46) and α-glucosidase SusB (GH97) may be responsible for the degradation of oligosaccharides to maltose. Thus, a full starch utilization system was found in all the Mariniflexile genomes except for M. gromovii KMM 6038T, which is consistent with the data from the biochemical tests.
Genomic data also confirmed the obtained biochemical characteristics (Table 2) that indicate that Mariniflexile spp., with the exception of M. maritimum KCTC 72895T, is not capable of agar hydrolyzing [1,4,8,10]. The gene encoding beta-agarase (EC 3.2.1.81) that shares 61% similarity to beta-agarase A (GH16, CAZ98338.1) of Zobellia galactanivorans DsijT [68] was found only in the M. maritimum KCTC 72895T genome.
Finally, we hypothesize that the environment can influence the gene composition of Mariniflexile species, providing them with an adaptive potential to degrade natural polysaccharides specific to the particular ecological niche. The most obvious difference between Mariniflexile strains isolated from the seashore or plants (KMM 9835T, Mariniflexile sp. TRM1-10, and M. soesokkakense KCTC 32427T) and from sea water or marine organisms (M. fucanivorans DSM 18792T, M. gromovii KCTC 12570T, and Mariniflexile sp. AS56) is that they have similar CAZomes, despite their distant positions on the phylogenomic tree (Figure S2); KMM 9835T is phylogenetically close to strain AS56 and most distant from the strains TRM1-10 and M. soesokkakense KCTC 32427T (Figure 5b).

3.4. Morphological, Physiological, and Biochemical Characteristics

Strain KMM 9835T was found to be Gram-negative, aerobic, non-motile bacteria. Colonies were yellow-pigmented shiny ones with regular edges of 2−3 mm in diameter on MA 2216. Electron microscopy observation revealed rod-shaped cells, 1.5−2.0 μm long and 0.7−0.9 μm in diameter, and extracellular material production was observed (Figure 6).
Phenotypic characteristics of strain KMM 9862T are given in Table 2 and Table 3, Figure S4, and in the species description. The bacterium KMM 9835T was able to grow in the narrow salinity range of 0–5% NaCl and at a temperature of 5–36 °C but was not able to utilize carbohydrates sources in the API 20E and API 20NE tests. It should be noted that the novel strain grew slowly without NaCl addition (0% NaCl) and weakly with 5% NaCl. The genomic DNA G+C content of strain KMM 9835T was 32.5 mol% (Table 2).
The dominant menaquinone was MK-6, and the major fatty acids were iso-C15:0, iso-C15:1 ω10c, and C15:0, followed by iso-C17:0 3-OH (Table 3). Fatty acid profiles were similar, with large proportions of iso-C15:0 and C15:0 found in all strains tested (Table 3). Strain KMM 9835T contained a slightly lower amount of anteiso-C15:0 2-OH, and M. maritimum KCTC 72895T contained a certain amount of iso-C14:0, iso-C16:1ω6c, iso-C16:0, and iso-C16:0 3-OH compared with other strains tested (Table 3).
The polar lipids of strain KMM 9835T consisted of phosphatidylethanolamine (PE), two unidentified aminolipids (AL1, AL2), an unidentified phospholipid (PL), and six unidentified lipids (L1-L6) (Figure S4). Strain KMM 9835T was close in its polar lipid profile to that of M. soesokkakense KCTC 32427T, except for the presence of PL, which was not found in the other two strains. The polar lipids of all strains tested included PE, AL1, AL2, and unidentified lipids L1-L6 or L1-L4, as in the case of M. maritimum KCTC 72895T. The latter additionally contained phosphatidylcholine, which was not present in other strains tested (Figure S4). The presence of iso-C15:0, iso-C15:1, C15:0, iso-C17:0 3-OH, and MK-6, and major polar lipid components, are corroborated with those previously described for Mariniflexile species [2,3,4,10].

4. Conclusions

The phylogenetic relationships observed on the basis of 16S rRNA gene and whole genome sequences and genetic distinctness as revealed by ANI and dDDH analyses were supported by phenotypic differences of the novel isolate KMM 9835T in its growth temperature and salinity ranges, enzyme activity, and substrate hydrolysis. Differential phenotypic characteristics are indicated in Table 2. Based on the combined phylogenetic evidence and phenotypic characteristics, it is proposed to classify marine sediment strain KMM 9835T as a novel species, Mariniflexile litorale sp. nov.
Description of Mariniflexile litorale sp. nov.
Mariniflexile litorale (li.to.ra’le. L. neut. adj. litorale, of the seashore, a shallow-seawater dweller).
Gram-negative, aerobic, oxidase-positive (weak reaction), catalase-positive, rod-shaped non-motile cells, 0.7–0.9 μm in width and 1.5–2.0 μm in length. Grows on MA 2216 or in MB 2216. On MA 2216, it produces hemi-transparent yellow-pigmented shiny smooth colonies with regular edges of 2–3 mm. Does not require NaCl for growth; growth occurs in 0–5 (w/v) NaCl with an optimum of 2% NaCl; growth in 0% and 5% NaCl is observed as slow and weak, respectively. The temperature range for growth is 5–36 °C, with an optimum of 25–28 °C. Does not grow at 4 °C and 37 °C. The pH range for growth is 5.5–9.5 (optimal pH 6.5–7.5). Positive for hydrolysis of starch, Tweens 20 and 40, and negative for hydrolysis of DNA, gelatin, casein, Tween 80, tyrosine, chitin, and agar, and production of H2S from thiosulfate. In the API 20E, negative for the ONPG test, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, H2S and urease production under anaerobic conditions, tryptophane deaminase, indole production, acetoin production (Voges-Proskauer reaction), gelatin hydrolysis, and oxidation/fermentation of D-sucrose, D-glucose, D-mannitol, inositol, D-sorbitol, L-rhamnose, D-melibiose, amygdalin, and L-arabinose. According to the API 20NE, positive for the PNPG test and esculin hydrolysis and negative for nitrate reduction, gelatin hydrolysis, indole production, glucose fermentation, arginine dihydrolase, urease, assimilation of D-glucose, D-mannitol, maltose, D-gluconate, L-malate assimilation of D-mannose, L-arabinose, N-acetylglucosamine, caprate, adipate, citrate, and phenylacetate.
Positive API ZYM test results are obtained for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase (weak reaction), acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-galactosidase, and N-acetyl-β-glucosaminidase, and negative for lipase (C14), trypsin, α-chymotrypsin, α-galactosidase, α-glucosidase, β-glucosidase, β-glucuronidase, α-mannosidase, and α-fucosidase.
The dominant menaquinone is MK-6, and the major fatty acids are iso-C15:0, iso-C15:1 ω10c, and C15:0. The polar lipids comprise phosphatidylethanolamine, two unidentified aminolipids, an unidentified phospholipid, and six unidentified lipids. The DNA GC content of 32.5% is calculated from the genome sequence.
The DDBJ/GenBank accession numbers for the 16S rRNA gene and genome sequences of strain KMM 9835T are OQ300347 and JASCRQ010000000 (GCF_031128465.2), respectively.
The type strain of the species is strain KMM 9835T (=KCTC 92792T), isolated from the sediment sample collected from the Amur Bay of the Sea of Japan seashore, Russia.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms12071413/s1, Figure S1: NJ/ML/MP tree based on 16S rRNA gene sequences available from the GenBank database showing relationships between the novel strain KMM 9835T (in bold), Mariniflexile species, and related taxa of the family Flavobacteriaceae. The NJ tree was reconstructed using the Kimura two-parameter model, and the ML tree was inferred under the GTR+GAMMA model. The branches are scaled in terms of the expected number of substitutions per site. The numbers above the branches represent bootstrap values with 1000 replicates larger than 60% (NJ/ML/MP). The bar indicates 0.02 accumulated substitutions per nucleotide position; Figure S2: ML tree based on concatenated sequences of 400 proteins showing the phylogenetic position of strains KMM 9835T, AS56, and TRM1-10 among Mariniflexile species and related taxa. Bootstrap values are based on 100 replicates. Bar, 0.20 substitutions per amino acid position; Figure S3: Pan-genome modeling. (a) Gene accumulation curves for the pan-genome (blue curve) and the core genome (green curve) of seven Mariniflexile genomes. Pan-genome curve: y = 2634.4x0.58 + 925.34. Core genome curve: y = 6139.3e−1.24x − 1780.27. (b) The new gene cluster number plot, curve: y = 1835.82x−0.49. Pan-genome openness was estimated under Heap’s law model [54]; Figure S4: Two-dimensional thin-layer chromatograms of polar lipids of strains: (a–c) KMM 9835T; (d–f) Mariniflexile maritimum KCTC 72895T; (g–i) Mariniflexile soesokkakense KCTC 32427T. Data were obtained from the present study. (a, d, g) non-specific detection of lipids prepared with 10% H2SO4 in methanol; (b,e,h) stained with ninhydrin; (c,f,i) stained with molybdate reagent. Abbreviations: PE, phosphatidylethanolamine; AL1, AL2, unidentified aminolipids; PL, unidentified phospholipid; AL, unidentified aminolipid; L1-L6, unidentified lipids.

Author Contributions

Investigation, L.R., E.B., Y.S., N.O., V.K., P.V. and M.I.; Methodology, L.R., N.O., V.K. and P.V.; Visualization, E.B., Y.S., N.O. and V.E.; Project administration, M.I.; Resources, M.I.; Software, N.O. and V.E.; Data curation, N.O. and M.I.; Writing—original draft, L.R., E.B., Y.S. and M.I.; Writing—review and editing, L.R. and M.I. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by a grant from the Ministry of Science and Higher Education, Russian Federation, 15.BRK.21.0004 (Contract No. 075-15-2021-1052).

Data Availability Statement

The type strain of the species is strain KMM 9835T (=KCTC 92792T), isolated from the sediment sample collected from the Amur Bay of the Sea of Japan seashore, Russia. The DDBJ/GenBank accession numbers for the 16S rRNA gene and genome sequences of strain KMM 9835T are OQ300347 and JASCRQ010000000, respectively. The DDBJ/GenBank accession number for the genome sequence of strain Mariniflexile soesokkakense KCTC 32427T is JAZHYP000000000.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Nedashkovskaya, O.I.; Kim, S.B.; Kwak, J.; Mikhailov, V.V.; Bae, K.S. Mariniflexile gromovii gen. nov., sp. nov., a gliding bacterium isolated from the sea urchin Strongylocentrotus intermedius. Int. J. Syst. Evol. Microbiol. 2006, 56, 1635–1638. [Google Scholar] [CrossRef] [PubMed]
  2. Jung, Y.T.; Kim, J.H.; Oh, T.K.; Yoon, J.H. Mariniflexile aquimaris sp. nov., isolated from seawater, and emended description of the genus Mariniflexile Nedashkovskaya et al. 2006. Int. J. Syst. Evol. Microbiol. 2012, 62, 539–544. [Google Scholar] [CrossRef] [PubMed]
  3. Jung, Y.T.; Yoon, J.H. Mariniflexile jejuense sp. nov., isolated from the junction between seawater and a freshwater spring, and emended description of the genus Mariniflexile. Int. J. Syst. Evol. Microbiol. 2013, 63, 1329–1334. [Google Scholar] [CrossRef] [PubMed]
  4. Park, S.; Jung, Y.T.; Yoon, J.H. Mariniflexile soesokkakense sp. nov., a member of the family Flavobacteriaceae isolated from the junction between the North Pacific Ocean and a freshwater spring, and emended description of the genus Mariniflexile. Antonie Van Leeuwenhoek 2013, 104, 769–777. [Google Scholar] [CrossRef] [PubMed]
  5. Garcia-Lopez, M.; Meier-Kolthoff, J.P.; Tindall, B.J.; Gronow, S.; Woyke, T.; Kyrpides, N.C.; Hahnke, R.L.; Goker, M. Analysis of 1000 Type-Strain Genomes Improves Taxonomic Classification of Bacteroidetes. Front. Microbiol. 2019, 10, 2083. [Google Scholar] [CrossRef] [PubMed]
  6. Oren, A.; Garrity, G.M. Valid publication of the names of forty-two phyla of prokaryotes. Int. J. Syst. Evol. Microbiol. 2021, 71, 005056. [Google Scholar] [CrossRef]
  7. Parte, A.C.; Sardà Carbasse, J.; Meier-Kolthoff, J.P.; Reimer, L.C.; Göker, M. List of Prokaryotic names with Standing in Nomenclature (LPSN) moves to the DSMZ. Int. J. Syst. Evol. Microbiol. 2020, 70, 5607–5612. [Google Scholar] [CrossRef] [PubMed]
  8. Ko, S.R.; Le, V.V.; Jin, L.; Lee, S.A.; Ahn, C.Y.; Oh, H.M. Mariniflexile maritimum sp. nov., isolated from seawater of the South Sea in the Republic of Korea. Int. J. Syst. Evol. Microbiol. 2021, 71, 4925. [Google Scholar] [CrossRef]
  9. Park, S.; Kang, C.H.; Park, J.M.; Yoon, J.H. Mariniflexile ostreae sp. nov., a member of the family Flavobacteriaceae isolated from an oyster. Antonie Van Leeuwenhoek 2014, 106, 555–563. [Google Scholar] [CrossRef]
  10. Barbeyron, T.; L’Haridon, S.; Michel, G.; Czjzek, M. Mariniflexile fucanivorans sp. nov., a marine member of the Flavobacteriaceae that degrades sulphated fucans from brown algae. Int. J. Syst. Evol. Microbiol. 2008, 58, 2107–2113. [Google Scholar] [CrossRef]
  11. Paillat, M.; Lunar Silva, I.; Cascales, E.; Doan, T. A journey with type IX secretion system effectors: Selection, transport, processing and activities. Microbiology 2023, 169, 001320. [Google Scholar] [CrossRef] [PubMed]
  12. McKee, L.S.; La Rosa, S.L.; Westereng, B.; Eijsink, V.G.; Pope, P.B.; Larsbrink, J. Polysaccharide degradation by the Bacteroidetes: Mechanisms and nomenclature. Environ. Microbiol. Rep. 2021, 13, 559–581. [Google Scholar] [CrossRef] [PubMed]
  13. Gerhardt, P.; Murray, R.G.E.; Wood, W.A.; Krieg, N.R. Methods for General and Molecular Bacteriology; American Society for Microbiology: Washington, DC, USA, 1994. [Google Scholar]
  14. Bowman, J.P. Description of Cellulophaga algicola sp. nov., isolated from the surface of Antarctic algae, and reclassification of Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Cellulophaga uliginosa comb. nov. Int. J. Syst. Evol. Microbiol. 2000, 50, 1861–1868. [Google Scholar] [CrossRef] [PubMed]
  15. Romanenko, L.A.; Tanaka, N.; Svetashev, V.I. Devosia submarina sp. nov., isolated from deep sea surface sediments. Int. J. Syst. Evol. Microbiol. 2013, 63, 3079–3085. [Google Scholar] [CrossRef] [PubMed]
  16. Romanenko, L.A.; Kurilenko, V.V.; Guzev, K.V.; Svetashev, V.I.; Mikhailov, V.V. Winogradskyella profunda sp. nov. isolated from the Chukchi Sea bottom sediment. Arch. Microbiol. 2019, 201, 45–50. [Google Scholar] [CrossRef] [PubMed]
  17. Folch, J.; Lees, M.; Sloane-Stanley, G.H. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 1957, 226, 497–509. [Google Scholar] [CrossRef] [PubMed]
  18. Collins, M.D.; Shah, H.N. Fatty acid, menaquinone and polar lipid composition of Rothia dentosacariosa. Arch. Microbiol. 1984, 137, 247–249. [Google Scholar] [CrossRef]
  19. Collins, M.D.; Goodfellow, M.; Minnikin, D.E. Fatty acid, isoprenoid quinone and polar lipid composition in the classification of Curtobacterium and related taxa. J. Gen. Microbiol. 1980, 118, 29–37. [Google Scholar] [CrossRef] [PubMed]
  20. Sasser, M. Identification of Bacteria by Gas Chromatography of Cellular Fatty Acids, MIDI Technical Note 101; MIDI Inc.: Newark, DE, USA, 1990. [Google Scholar]
  21. Svetashev, V.I. Mild method for preparation of 4,4-dimethyloxazoline derivatives of polyunsaturated fatty acids for GC-MS. Lipids 2011, 46, 463–467. [Google Scholar] [CrossRef]
  22. Hiraishi, A.; Ueda, Y.; Ishihara, J.; Mori, T. Comparative lipoquinone analysis of influent sewage and activated sludge by high-performance liquid chromatography and photodiode array detection. J. Gen. Appl. Microbiol. 1996, 42, 457–459. [Google Scholar] [CrossRef]
  23. Fautz, E.; Reichenbach, H. A simple test for flexirubin-type pigments. FEMS Microbiol. Lett. 1980, 8, 87–91. [Google Scholar] [CrossRef]
  24. Yoon, S.H.; Ha, S.M.; Kwon, S.; Lim, J.; Kim, Y.; Seo, H.; Chun, J. Introducing EzBioCloud: A taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int. J. Syst. Evol. Microbiol. 2017, 67, 1613. [Google Scholar] [CrossRef]
  25. Meier-Kolthoff, J.P.; Carbasse, J.S.; Peinado-Olarte, R.L.; Göker, M. TYGS and LPSN: A database tandem for fast and reliable genome-based classification and nomenclature of prokaryotes. Nucleic Acids Res. 2022, 7, D801–D807. [Google Scholar] [CrossRef] [PubMed]
  26. Meier-Kolthoff, J.P.; Hahnke, R.L.; Petersen, J.; Scheuner, C.; Michael, V.; Fiebig, A.; Rohde, C.; Rohde, M.; Fartmann, B.; Goodwin, L.A.; et al. Complete genome sequence of DSM 30083T, the type strain (U5/41T) of Escherichia coli, and a proposal for delineating subspecies in microbial taxonomy. Stand. Genom. Sci. 2014, 8, 10. [Google Scholar] [CrossRef]
  27. Stamatakis, A. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 2014, 30, 1312–1313. [Google Scholar] [CrossRef]
  28. Goloboff, P.A.; Farris, J.S.; Nixon, K.C. TNT, a free program for phylogenetic analysis. Cladistics 2008, 24, 774–786. [Google Scholar] [CrossRef]
  29. Tamura, K.; Stecher, G.; Kumar, S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol. Biol. Evol. 2021, 38, 3022–3027. [Google Scholar] [CrossRef] [PubMed]
  30. Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 2014, 15, 2114–2120. [Google Scholar] [CrossRef]
  31. Bankevich, A.; Nurk, S.; Antipov, D.; Gurevich, A.A.; Dvorkin, M.; Kulikov, A.S.; Lesin, V.M.; Nikolenko, S.I.; Pham, S.; Prjibelski, A.D.; et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 2012, 19, 455–477. [Google Scholar] [CrossRef]
  32. Gurevich, A.; Saveliev, V.; Vyahhi, N.; Tesler, G. QUAST: Quality assessment tool for genome assemblies. Bioinformatics 2013, 29, 1072–1075. [Google Scholar] [CrossRef]
  33. Wick, R.R.; Judd, L.M.; Gorrie, C.L.; Holt, K.E. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput. Biol. 2017, 13, e1005595. [Google Scholar] [CrossRef] [PubMed]
  34. Danecek, P.; Bonfield, J.K.; Liddle, J.; Marshall, J.; Ohan, V.; Pollard, M.O.; Whitwham, A.; Keane, T.; McCarthy, S.A.; Davies, R.M.; et al. Twelve years of SAMtools and BCFtools. GigaScience 2021, 10, giab008. [Google Scholar] [CrossRef] [PubMed]
  35. Parks, D.H.; Imelfort, M.; Skennerton, C.T.; Hugenholtz, P.; Tyson, G.W. CheckM: Assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 2015, 25, 1043–1055. [Google Scholar] [CrossRef] [PubMed]
  36. Rodriguez-R, L.M.; Konstantinidis, K.T. The enveomics collection: A toolbox for specialized analyses of microbial genomes and metagenomes. PeerJ Prepr. 2016, 4, e1900v1. [Google Scholar] [CrossRef]
  37. Meier-Kolthoff, J.P.; Göker, M. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat. Commun. 2019, 10, 2182. [Google Scholar] [CrossRef] [PubMed]
  38. Asnicar, F.; Thomas, A.M.; Beghini, F.; Mengoni, C.; Manara, S.; Manghi, P.; Zhu, Q.; Bolzan, M.; Cumbo, F.; May, U.; et al. Precise phylogenetic analysis of microbial isolates and genomes from metagenomes using PhyloPhlAn 3.0. Nat. Commun. 2020, 11, 2500. [Google Scholar] [CrossRef] [PubMed]
  39. Tatusova, T.; DiCuccio, M.; Badretdin, A.; Chetvernin, V.; Nawrocki, E.P.; Zaslavsky, L.; Lomsadze, A.; Pruitt, K.D.; Borodovsky, M.; Ostell, J. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 2016, 44, 6614–6624. [Google Scholar] [CrossRef] [PubMed]
  40. Aziz, R.K.; Bartels, D.; Best, A.A.; DeJongh, M.; Disz, T.; Edwards, R.A.; Formsma, K.; Gerdes, S.; Glass, E.M.; Kubal, M.; et al. The RAST Server: Rapid annotations using subsystems technology. BMC Genom. 2008, 9, 75. [Google Scholar] [CrossRef] [PubMed]
  41. Seemann, T. Prokka: Rapid prokaryotic genome annotation. Bioinformatics 2014, 30, 2068–2069. [Google Scholar] [CrossRef]
  42. Grant, J.R.; Enns, E.; Marinier, E.; Mandal, A.; Herman, E.K.; Chen, C.-Y.; Graham, M.; Van Domselaar, G.; Stothard, P. Proksee: In-Depth Characterization and Visualization of Bacterial Genomes. Nucleic Acids Res. 2023, 51, 484–492. [Google Scholar] [CrossRef]
  43. Vernikos, G.S.; Parkhill, J. Interpolated variable order motifs for identification of horizontally acquired DNA: Revisiting the Salmonella pathogenicity islands. Bioinformatics 2006, 22, 2196–2203. [Google Scholar] [CrossRef] [PubMed]
  44. Couvin, D.; Bernheim, A.; Toffano-Nioche, C.; Touchon, M.; Michalik, J.; Néron, B.; Rocha, E.P.C.; Vergnaud, G.; Gautheret, D.; Pourcel, C. CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins. Nucleic Acids Res. 2018, 46, 246–251. [Google Scholar] [CrossRef] [PubMed]
  45. Dong, M.J.; Luo, H.; Gao, F. Ori-Finder 2022: A Comprehensive Web Server for Prediction and Analysis of Bacterial Replication Origins. GPB 2022, 6, 1207–1213. [Google Scholar] [CrossRef] [PubMed]
  46. Neron, B.; Denise, R.; Coluzzi, C.; Touchon, M.; Rocha, E.P.; Abby, S.S. MacSyFinder v2: Improved modelling and search engine to identify molecular systems in genomes. Peer Community J. 2023, 3, e28. [Google Scholar] [CrossRef]
  47. Abby, S.S.; Cury, J.; Guglielmini, J.; Néron, B.; Touchon, M.; Rocha, E.P. Identification of protein secretion systems in bacterial genomes. Sci. Rep. 2016, 6, 23080. [Google Scholar] [CrossRef] [PubMed]
  48. Zheng, J.; Ge, Q.; Yan, Y.; Zhang, X.; Huang, L.; Yin, Y. dbCAN3: Automated carbohydrate-active enzyme and substrate annotation. Nucleic Acids Res. 2023, 51, 115–121. [Google Scholar] [CrossRef] [PubMed]
  49. Ausland, C.; Zheng, J.; Yi, H.; Yang, B.; Li, T.; Feng, X.; Zheng, B.; Yin, Y. dbCAN-PUL: A database of experimentally characterized CAZyme gene clusters and their substrates. Nucleic Acids Res. 2021, 49, D523–D528. [Google Scholar] [CrossRef] [PubMed]
  50. Eren, A.M.; Esen, O.C.; Quince, C.; Vineis, J.H.; Morrison, H.G.; Sogin, M.L.; Delmont, T.O. Anvi’o: An advanced analysis and visualization platformfor ‘omics data. PeerJ 2015, 3, e1319. [Google Scholar] [CrossRef] [PubMed]
  51. Kanehisa, M.; Goto, S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000, 28, 27–30. [Google Scholar] [CrossRef]
  52. Moriya, Y.; Itoh, M.; Okuda, S.; Yoshizawa, A.; Kanehisa, M. KAAS: An automatic genome annotation and pathway reconstruction server. Nucleic Acids Res. 2007, 35, W182–W185. [Google Scholar] [CrossRef]
  53. Zhao, Y.; Jia, X.; Yang, J.; Ling, Y.; Zhang, Z.; Yu, J.; Wu, J.; Xiao, J. PanGP: A tool for quickly analyzing bacterial pan-genome profile. Bioinformatics 2014, 30, 1297–1299. [Google Scholar] [CrossRef] [PubMed]
  54. Tettelin, H.; Riley, D.; Cattuto, C.; Medini, D. Comparative genomics: The bacterial pan-genome. Curr. Opin. Microbiol. 2008, 11, 472–477. [Google Scholar] [CrossRef] [PubMed]
  55. Kim, M.; Oh, H.S.; Park, S.C.; Chun, J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int. J. Syst. Evol. Microbiol. 2014, 64, 346–351. [Google Scholar] [CrossRef] [PubMed]
  56. Richter, M.; Rosselló-Móra, R. Shifting the genomic gold standard for the prokaryotic species definition. Proc. Natl. Acad. Sci. USA 2009, 106, 19126–19131. [Google Scholar] [CrossRef] [PubMed]
  57. Goris, J.; Konstantinidis, K.T.; Klappenbach, J.A.; Coenye, T.; Vandamme, P.; Tiedje, J.M. DNA–DNA hybridization values and their relationship to whole-genome sequence similarities. Int. J. Syst. Evol. Microbiol. 2007, 57, 81–91. [Google Scholar] [CrossRef] [PubMed]
  58. Chun, J.; Oren, A.; Ventosa, A.; Christensen, H.; Arahal, D.R.; Da Costa, M.S.; Rooney, A.P.; Yi, H.; Xu, X.W.; De Meyer, S.; et al. Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int. J. Syst. Evol. Microbiol. 2018, 68, 461–468. [Google Scholar] [CrossRef] [PubMed]
  59. Riesco, R.; Trujillo, M.E. Update on the proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int. J. Syst. Evol. Microbiol. 2024, 74, 006300. [Google Scholar] [CrossRef]
  60. Jordan, D.B.; Wagschal, K.; Grigorescu, A.A.; Braker, J.D. Highly active β-xylosidases of glycoside hydrolase family 43 operating on natural and artificial substrates. Appl. Microbiol. Biotechnol. 2013, 97, 4415–4428. [Google Scholar] [CrossRef]
  61. Pijning, T.; van Pouderoyen, G.; Kluskens, L.; van der Oost, J.; Dijkstra, B.W. The crystal structure of a hyperthermoactive exopolygalacturonase from Thermotoga maritima reveals a unique tetramer. FEBS Lett. 2009, 583, 3665–3670. [Google Scholar] [CrossRef]
  62. Zeugner, L.E.; Krüger, K.; Barrero-Canosa, J.; Amann, R.I.; Fuchs, B.M. In situ visualization of glycoside hydrolase family 92 genes in marine flavobacteria. ISME Commun. 2021, 1, 81. [Google Scholar] [CrossRef]
  63. Nakamura, A.M.; Nascimento, A.S.; Polikarpov, I. Structural diversity of carbohydrate esterases. Biotechnol. Res. Innov. 2017, 1, 35–51. [Google Scholar] [CrossRef]
  64. Ameri, R.; García, J.L.; Derenfed, A.B.; Pradel, N.; Neifar, S.; Mhiri, S.; Mezghanni, M.; Jaouadi, N.Z.; Barriuso, J.; Bejar, S. Genome sequence and Carbohydrate Active Enzymes (CAZymes) repertoire of the thermophilic Caldicoprobacter algeriensis TH7C1T. Microb. Cell Fact. 2022, 21, 91. [Google Scholar] [CrossRef] [PubMed]
  65. Chen, B.; Liu, G.; Chen, Q.; Wang, H.; Liu, L.; Tang, K. Discovery of a novel marine Bacteroidetes with a rich repertoire of carbohydrate-active enzymes. Comput. Struct. Biotechnol. J. 2023, 23, 406–416. [Google Scholar] [CrossRef]
  66. Lasica, A.M.; Ksiazek, M.; Madej, M.; Potempa, J. The Type IX Secretion System (T9SS): Highlights and Recent Insights into Its Structure and Function. Front. Cell Infect. Microbiol. 2017, 7, 215. [Google Scholar] [CrossRef] [PubMed]
  67. Foley, M.H.; Cockburn, D.W.; Koropatkin, N.M. The Sus operon: A model system for starch uptake by the human gut Bacteroidetes. Cell Mol. Life Sci. 2016, 73, 2603–2617. [Google Scholar] [CrossRef]
  68. Thomas, F.; Barbeyron, T.; Tonon, T.; Génicot, S.; Czjzek, M.; Michel, G. Characterization of the first alginolytic operons in a marine bacterium: From their emergence in marine Flavobacteriia to their independent transfers to marine Proteobacteria and human gut Bacteroides. Environ. Microbiol. 2012, 9, 2379–2394. [Google Scholar] [CrossRef]
Microorganisms 12 01413 g001
Figure 1. NJ/ML/MP tree based on 16S rRNA gene sequences available from the GenBank database showing relationships between the novel strain KMM 9835T (in bold), Mariniflexile species, and related taxa of the family Flavobacteriaceae. The NJ tree was reconstructed using the Kimura two-parameter model. The ML tree was inferred under the GTR + GAMMA model. The branches are scaled in terms of the expected number of substitutions per site. The numbers above the branches represent bootstrap values with 1000 replicates larger than 60% (NJ/ML/MP). The bar indicates 0.02 accumulated substitutions per nucleotide position.
Figure 1. NJ/ML/MP tree based on 16S rRNA gene sequences available from the GenBank database showing relationships between the novel strain KMM 9835T (in bold), Mariniflexile species, and related taxa of the family Flavobacteriaceae. The NJ tree was reconstructed using the Kimura two-parameter model. The ML tree was inferred under the GTR + GAMMA model. The branches are scaled in terms of the expected number of substitutions per site. The numbers above the branches represent bootstrap values with 1000 replicates larger than 60% (NJ/ML/MP). The bar indicates 0.02 accumulated substitutions per nucleotide position.
Microorganisms 12 01413 g001
Microorganisms 12 01413 g002
Figure 2. ML tree based on concatenated sequences of 341 translated proteins showing the phylogenetic position of strain KMM 9835T among Mariniflexile species and related taxa. The tree was inferred under the PROTCATLG evolutionary model using 100 replicates for bootstrapping. Bar: 0.20 substitutions per amino acid position.
Figure 2. ML tree based on concatenated sequences of 341 translated proteins showing the phylogenetic position of strain KMM 9835T among Mariniflexile species and related taxa. The tree was inferred under the PROTCATLG evolutionary model using 100 replicates for bootstrapping. Bar: 0.20 substitutions per amino acid position.
Microorganisms 12 01413 g002
Microorganisms 12 01413 g003
Figure 3. Chromosome map of strain KMM 9835T created using the Proksee server [42]. The scale is shown in megabases (Mbp) on the inside circle. Starting with the inner rings, the first two circles represent GC content (in black) and GC skew (G−C)/(G+C) (in violet blue and light green). The next two dark red circles show reverse and forward strand CDSs. Moving outward, the dark green circle shows PULs designated as CGCs, annotated by the dbCAN server [49]. The outermost circle shows the CRISPR-Cas region (in black). The figure also shows retron-type RNA-directed DNA polymerase (EC 2.7.7.49) (designated as retron 1–13 with black labels), rrn operons (blue labels), oriC (leuB_2 and dnaA), and ter (mnmG) (red labels).
Figure 3. Chromosome map of strain KMM 9835T created using the Proksee server [42]. The scale is shown in megabases (Mbp) on the inside circle. Starting with the inner rings, the first two circles represent GC content (in black) and GC skew (G−C)/(G+C) (in violet blue and light green). The next two dark red circles show reverse and forward strand CDSs. Moving outward, the dark green circle shows PULs designated as CGCs, annotated by the dbCAN server [49]. The outermost circle shows the CRISPR-Cas region (in black). The figure also shows retron-type RNA-directed DNA polymerase (EC 2.7.7.49) (designated as retron 1–13 with black labels), rrn operons (blue labels), oriC (leuB_2 and dnaA), and ter (mnmG) (red labels).
Microorganisms 12 01413 g003
Microorganisms 12 01413 g004
Figure 4. The pan-genome of seven strains of Mariniflexile spp. generated with anvi’o [50]. Circle bars represent the presence/absence of 9163 pan-genomic clusters in each genome. Gene clusters are organized as core (green), shell (yellow), cloud (red), and singleton (purple) gene clusters using Euclidian distance and Ward ordination. The heatmap in the upper right corner shows pairwise values of average nucleotide identity (ANI) in percentages. The bars under the heatmap show, relative to each genome, the number of gene clusters (0–3881), number of singleton gene clusters (0–1010), GC-content (0–0.37778), and total length (0–4,858,325). The strain KMM 9835T is colored red. Other information included in the figure comprises the maximum number of paralogs, combined homogeneity index, single-copy gene clusters (SCG clusters), and KOfam and KEGG modules (green and light green circles).
Figure 4. The pan-genome of seven strains of Mariniflexile spp. generated with anvi’o [50]. Circle bars represent the presence/absence of 9163 pan-genomic clusters in each genome. Gene clusters are organized as core (green), shell (yellow), cloud (red), and singleton (purple) gene clusters using Euclidian distance and Ward ordination. The heatmap in the upper right corner shows pairwise values of average nucleotide identity (ANI) in percentages. The bars under the heatmap show, relative to each genome, the number of gene clusters (0–3881), number of singleton gene clusters (0–1010), GC-content (0–0.37778), and total length (0–4,858,325). The strain KMM 9835T is colored red. Other information included in the figure comprises the maximum number of paralogs, combined homogeneity index, single-copy gene clusters (SCG clusters), and KOfam and KEGG modules (green and light green circles).
Microorganisms 12 01413 g004
Microorganisms 12 01413 g005
Figure 5. Distribution of CAZymes within the Mariniflexile genus. (a) Number of CAZyme classes in KMM 9835T and other Mariniflexile species. (b) Heatmap of CAZyme family abundance in Mariniflexile species. GH—glycoside hydrolase, GT—glycosyltransferase, CE—carbohydrate esterase, PL—polysaccharide lyase, AA—auxiliary activity.
Figure 5. Distribution of CAZymes within the Mariniflexile genus. (a) Number of CAZyme classes in KMM 9835T and other Mariniflexile species. (b) Heatmap of CAZyme family abundance in Mariniflexile species. GH—glycoside hydrolase, GT—glycosyltransferase, CE—carbohydrate esterase, PL—polysaccharide lyase, AA—auxiliary activity.
Microorganisms 12 01413 g005
Microorganisms 12 01413 g006
Figure 6. A transmission electron micrograph of strain KMM 9835T, grown on MA 2216. Bar, 1 µm.
Figure 6. A transmission electron micrograph of strain KMM 9835T, grown on MA 2216. Bar, 1 µm.
Microorganisms 12 01413 g006
Table 1. Genomic features of strains KMM 9835T, KCTC 32427T, and other Mariniflexile strains.
Table 1. Genomic features of strains KMM 9835T, KCTC 32427T, and other Mariniflexile strains.
Feature1234567
Assembly levelchromosomecontigscaffoldscaffoldscaffoldchromosomecontig
Genome size (Mb)4.53.74.74.33.74.94.7
Number of contigs127295238150
G+C Content (mol%)32.533.53333.537.534.534
N50 (Kb)4521.4648.1432.9177.4292.64858.3288.3
L501347416
Coverage180.0×63.0×317.0×100.0×100.0×219.1×207.0×
Total genes3839315841043770315739873789
Protein coding genes3771310039923690308839153722
rRNAs (5S/16S/23S)2/2/21/1/11/1/11/1/11/1/12/2/21/1/1
tRNA39364037384137
checkM completeness (%)10010099.3598.22100100100
checkM contamination (%)0.810.321.050.491.290.320.97
WGS project/RefSeqGCF_031128465.2JAZHYP01SLUP01JAGJCB01WRPN01NZ_CP022985JAUPED01
Genome assemblyASM3112846v2ASM3965036v1ASM434123v1ASM1781443v1ASM974656v1ASM342598v1ASM3055247v1
Strains: 1, KMM 9835T; 2, M. soesokkakense KCTC 32427T; 3, M. fucanivorans DSM 18792T; 4, M. gromovii KCTC 12570T; 5, M. maritimum M5A1MT; 6, Mariniflexile sp. TRM1-10; 7, Mariniflexile sp. AS56.
Table 2. Differential characteristics of strain 9835T and the type strains of the most closely related Mariniflexile species.
Table 2. Differential characteristics of strain 9835T and the type strains of the most closely related Mariniflexile species.
Characteristic12345
DNA GC content (%) *32.533.537.533.533.0
Growth in:
5% NaCl(+)−(+)++
6% NaCl−−−+−
Growth at:
5 °C+−−++
36 °C(+)(+)++−
37 °C−−++−
Oxidase reaction(+)++−−
Hydrolysis of:
Starch+++−+
Agar−−+−−
Gelatin−−−+−
DNA−−+(+)+
Tween-40+−−−−
API ZYM tests:
Esterase C4+(+)+++
Lipase C 14−−−−(+)
Valine arylamidase+−+++
Cystine arylamidase(+)−(+)++
Trypsin−−−++
α-chymotrypsin−−−++
α-galactosidase−++−−
α-glucosidase−++−+
β-glucosidase−−+−+
α-fucosidase−−−−+
Strains: 1, KMM 9835T; 2, M. soesokkakense KCTC 32427T (data were obtained from the present study unless otherwise indicated); 3, M. maritimum KCTC 72895T (data were obtained from the present study unless otherwise indicated); 4, M. gromovii KMM 6038T (data from Nedashkovskaya et al., 2006 [1]; Barbeyron et al., 2008 [10]); 5, M. fucanivorans DSM 18792T (data from Barbeyron et al., 2008 [10]). * The DNA GC contents of the strains KMM 9835T, M. maritimum KCTC 72895T, M. gromovii KCTC 12570T, and M. fucanivorans DSM 18792T were derived from the GenBank. +, positive; −, negative; (+), weak reaction. All strains were positive for hydrolysis of aesculin, alkaline phosphatase, esterase lipase C8, leucine arylamidase, acid phosphatase, and β-galactosidase activities, and negative for production of flexirubin pigments, α-mannosidase, and β-glucuronidase.
Table 3. Cellular fatty acid composition (%) of strain KMM 9835T and type strains of related Mariniflexile species.
Table 3. Cellular fatty acid composition (%) of strain KMM 9835T and type strains of related Mariniflexile species.
Fatty Acid123
iso-C14:00.543.260.73
iso-C15:1 ω10c12.743.979.80
iso-C15:012.8715.5116.96
anteiso-C15:05.316.345.71
C15:1 ω11c2.131.421.91
C15:1 ω6c4.203.035.25
C15:011.407.5912.54
iso-C16:1 ω6c0.374.350.47
iso-C16:00.584.050.62
C16:1 ω7c4.423.712.82
iso-C15:0 2-OH3.264.701.81
anteiso-C15:0 2-OH6.021.771.81
C16:00.881.580.81
iso-C15:0 3-OH6.444.6710.31
anteiso-C15:0 3-OH1.582.591.72
iso-C17:1 ω7c1.271.170.73
C15:0 3-OH2.861.422.73
C17:1 ω6c1.101.521.49
C16:0 2-OH0.071.580.11
iso-C16:0 3-OH3.3611.454.42
anteiso-C16:0 3-OH1.061.531.20
iso-C17:0 3-OH9.855.509.50
anteiso-C17:0 3-OH1.281.111.15
Strains: 1, KMM 9835T; 2, M. maritimum KCTC 72895T; 3, M. soesokkakense KCTC 32427T (data were obtained from the present study).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Romanenko, L.; Bystritskaya, E.; Savicheva, Y.; Eremeev, V.; Otstavnykh, N.; Kurilenko, V.; Velansky, P.; Isaeva, M. Description and Whole-Genome Sequencing of Mariniflexile litorale sp. nov., Isolated from the Shallow Sediments of the Sea of Japan. Microorganisms 2024, 12, 1413. https://doi.org/10.3390/microorganisms12071413

AMA Style

Romanenko L, Bystritskaya E, Savicheva Y, Eremeev V, Otstavnykh N, Kurilenko V, Velansky P, Isaeva M. Description and Whole-Genome Sequencing of Mariniflexile litorale sp. nov., Isolated from the Shallow Sediments of the Sea of Japan. Microorganisms. 2024; 12(7):1413. https://doi.org/10.3390/microorganisms12071413

Chicago/Turabian Style

Romanenko, Lyudmila, Evgeniya Bystritskaya, Yuliya Savicheva, Viacheslav Eremeev, Nadezhda Otstavnykh, Valeriya Kurilenko, Peter Velansky, and Marina Isaeva. 2024. "Description and Whole-Genome Sequencing of Mariniflexile litorale sp. nov., Isolated from the Shallow Sediments of the Sea of Japan" Microorganisms 12, no. 7: 1413. https://doi.org/10.3390/microorganisms12071413

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop