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Article

Muricauda okinawensis sp. Nov. and Muricauda yonaguniensis sp. Nov., Two Marine Bacteria Isolated from the Sediment Core near Hydrothermal Fields of Southern Okinawa Trough

by
Wenrui Cao
1,*,
Xingyu Deng
1,2,
Mingyu Jiang
1,
Zhigang Zeng
1 and
Fengming Chang
1
1
Key Laboratory of Marine Geology and Environment, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
2
College of Earth Science and Engineering, Shandong University of Science and Technology, Qingdao 266590, China
*
Author to whom correspondence should be addressed.
Microorganisms 2023, 11(6), 1580; https://doi.org/10.3390/microorganisms11061580
Submission received: 25 May 2023 / Revised: 9 June 2023 / Accepted: 13 June 2023 / Published: 14 June 2023
(This article belongs to the Section Systems Microbiology)
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Abstract

:
Two strains, 81s02T and 334s03T, were isolated from the sediment core near the hydrothermal field of southern Okinawa Trough. The cells of both strains were observed to be rod-shaped, non-gliding, Gram-staining negative, yellow-pigmented, facultatively anaerobic, catalase and oxidase positive, and showing optimum growth at 30 °C and pH 7.5. The strains 81s02T and 334s03T were able to tolerate up to 10% and 9% (w/v) NaCl concentration, respectively. Based on phylogenomic analysis, the average nucleotide identity (ANI) and the digital DNA-DNA hybridization (dDDH) values between the two strains and the nearest phylogenetic neighbors of the genus Muricauda were in range of 78.0–86.3% and 21.5–33.9%, respectively. The strains 81s02T and 334s03T shared 98.1% 16S rRNA gene sequence similarity to each other but were identified as two distinct species based on 81.4–81.5% ANIb, 85.5–85.6% ANIm and 25.4% dDDH values calculated using whole genome sequences. The strains 81s02T and 334s03T shared the highest 16S rRNA gene sequence similarity to M. lutimaris SMK-108T (98.7%) and M. aurea BC31-1-A7T (98.8%), respectively. The major fatty acid of strains 81s02T and 334s03T were identified similarly as iso-C15:0, iso-C17:0 3-OH and iso-C15:1 G, and the major polar lipids of the both strains consisted of phosphatidylethanolamine and two unidentified lipids. The strains contained MK-6 as their predominant menaquinone. The genomic G+C contents of strains 81s02T and 334s03T were determined to be 41.6 and 41.9 mol%, respectively. Based on the phylogenetic and phenotypic characteristics, both strains are considered to represent two novel species of the genus Muricauda, and the names Muricauda okinawensis sp. nov. and Muricauda yonaguniensis sp. nov. are proposed for strains 81s02T (=KCTC 92889T = MCCC 1K08502T) and 334s03T (=KCTC 92890T = MCCC 1K08503T).

1. Introduction

The genus Muricauda, a member of the family Flavobacteriaceae within the class Flavobacteriia, was first proposed by Bruns et al. [1], with Muricauda ruestringensis as the type species, and subsequently emended by Yoon et al. [2], Hwang et al. [3] and Liang et al. [4]. Consequently, Flagellimonas and Spongiibacterium were transferred to genus Muricauda [4,5], and Flagellimonas algicola [6], Flagellimonas pacifica [7,8], Flagellimonas maritima [9], Flagellimonas aquimarina [8], Flagellimonas eckloniae [10] and Spongiibacterium flavum [8,11] were reclassified as Muricauda algicola [4], Muricauda parva [4], Muricauda aurantiaca [4], Muricauda koreensis [5], Muricauda eckloniae [5] and Muricauda flava [5], respectively. On the other hand, one species of genus Muricauda, named Muricauda lutea [12], was reclassified as Croceivirga lutea [13]. At present, the genus Muricauda comprises 37 species with valid published and correct names and two species with not validly published names (https://lpsn.dsmz.de/genus/muricauda, accessed on 1 May 2023). Most of the these species were isolated from marine environments, such as seawater [3,14,15,16,17], tidal flats [18,19,20], coast sand [21], mangrove wetland [22], deep marine sediments [23,24,25], marine plants [6,8,10] and marine animals [11,26,27,28]. Members of genus Muricauda were characterized as Gram-stain negative, rod-shaped and having a DNA G+C content of 38–57 mol%, with MK-6 as the major isoprenoid quinone.
The deep-sea hydrothermal field represents one of the most physically and chemically diverse biomes on the Earth [29,30]. The Okinawa Trough (OT) contains several active hydrothermal fields, altogether making it an interesting region. A few strains had been isolated from the hydrothermal fields of the OT, where the composition of prokaryotic communities in different regions was also investigated [31,32,33,34,35,36,37,38]. It was suggested that Muricauda had never been the main group of microorganisms in the seafloor hydrothermal fields, although it did exist in the environment. In this study, during the investigation of the bacterial community from the sediment core near the hydrothermal fields of southern OT, two strains (designated 81s02T and 334s03T), representing two novel members of the genus Muricauda, were characterized after isolation and purification via polyphasic taxonomy and comparative genome analysis.

2. Materials and Methods

2.1. Sampling, Isolation and Maintenance

The samples were recovered by a gravity corer from southern OT during the HOBAB4 cruise of the R/V Kexue at Station S2 in 2016. Sediment core HOBAB4-S2 (24°52′49.91″ N; 122°37′19.70″ E; water depth, 1505 m) was collected from a rifted basin between the Yonaguni Knoll IV and Tangyin hydrothermal field. Marine sediment dilutions (up to 10−2, 0.1 g sediment in 9.9 mL artificial sea water) were spread-plated on marine agar 2216 (MA, pH 7.2; BD Difco, New York, NY, USA) [39] and incubated at 25 °C under aerobic condition. Two isolates, designated as 81s02 and 334s03, were obtained after being incubated for 7 days from the samples of 81 cm and 334 cm below surface, respectively. The strains were stored at −80 °C in 20% (v/v) glycerol. The type strains Muricauda ruestringensis B1T (=DSM 13258T), Muricauda aurea BC31-1-A7T (=MCCC M23246T), Muricauda aquimarina SW-63T (=JCM 11811T) and Muricauda lutimaris SMK-108T (=KCTC 22173T), purchased from the Marine Culture Collection of China (MCCC), were used as reference strains for comparative purposes. Unless otherwise described, the strains were cultivated on marine agar 2216 (MA; Difco) at pH 7.5 and 30 °C.

2.2. 16S rRNA Gene Sequence and Phylogeny

Genomic DNA was extracted using a genomic DNA extraction kit (TIANGEN Biotech Co., Ltd., Beijing, China) according to the manufacturer’s instructions. The 16S rRNA gene was amplified by the universal primers 27F and 1492R, as previously described [40]. The PCR product was purified and ligated into the PMD19-T vector (TaKaRa, Kusatsu, Japan) and cloned according to the manufacturer’s instructions. Sequencing was performed by BGI (Qingdao, China). A BLAST search (https://www.ncbi.nlm.nih.gov, accessed on 10 April 2023) and the EzTaxon-e server (http://www.ezbiocloud.net, accessed on 10 April 2023) were used to calculate the pairwise sequence similarity based on the almost complete 16S rRNA gene sequences [41]. The multiple alignments of sequences for the new strains (81s02T and 334s03T) and other type strains of the most closely related species were performed using CLUSTAL X [42]. A phylogenetic analysis was subsequently performed and the phylogenetic trees were constructed with MEGA version X [43] using the neighbor-joining (NJ) [44], maximum parsimony (MP) [45] and maximum likelihood (ML) [46] algorithms. The bootstrap analyses were based on 1000 re-samplings and the complete deletion option was used for the analysis [47], and the distances were calculated according to the two-parameter model of Kimura [48].

2.3. Genomic Characterization

The draft genomes of strains 81s02T and 334s03T were sequenced using Allwegene Tech Co. Ltd. (Beijing, China), using the Illumina HiSeq platform. A raw sequencing data assembly was performed using SOAPdenovo [49,50]. Open Reading Frames (ORFs) were predicted using prodigal (v2.6.3) [51] and the identification of tRNAs and rRNAs was carried out using tRNAscan-SE (v1.3.1) [52]. Protein coding regions were annotated against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [53]. The assignments of key metabolic pathways and specific functions were manually verified based on the KEGG result and the online KEGG mapping tools (https://www.genome.jp/kegg/kegg1b.html, accessed on 12 May 2023). The functional annotation of the genomes was also performed with rapid annotations using subsystems technology (RAST) to estimate genes involved in different categories (https://rast.nmpdr.org/, accessed on 12 May 2023) [54,55]. The Carbohydrate-Active Enzymes database (http://www.cazy.org/, accessed on 13 May 2023) [56] was used to predict carbohydrate-active enzymes. To classify the position of 81s02T and 334s03T in the genus Muricauda using genome sequence-based comparison, the digital DNA–DNA hybridization (dDDH) value was calculated using the Genome-to-Genome Distance Calculator 3.0 (http://ggdc.dsmz.de/ggdc.php/, accessed on 26 April 2023) with the recommended parameter, Formula 2 [57]. Average nucleotide identity calculation based on Blast+ (ANIb) and MUMmer (ANIm) was estimated using JspeciesWS (http://jspecies.ribohost.com/jspeciesws/, accessed on 24 May 2023) [58]. The draft genome sequences of strain M. abyssi W52T (JAHZSV000000000), M. aquimarina SW-63T (RZMZ00000000), M. aurea BC31-1-A7T (JAFLNL000000000), M. brasiliensis K001T (QBTW00000000), M. chongwuensis HICWT (WYET00000000), M. lutimaris SMK-108T (QXFH00000000), M. oceanensis 40DY170T (RZNA00000000), M. oceani 501str8T (CP049616) and M. ruestringensis B1T (CP002999) were obtained from the GenBank database for the overall genome-related index (OGRI) analyses [59]. The draft genomes of two Muricauda strains in this study and close relatives affiliated with the genus Muricauda were downloaded from the NCBI GenBank database. The 120 conserved concatenated proteins (Bac120 sets) were identified using GTDB-Tk v. 1.3.0 and used to construct a phylogenetic tree using FastTree [60,61,62]. Formosa maritima was used as an outgroup.

2.4. Morphological, Physiological and Biochemical Characterization

Cell morphology was observed using the 3-day cultures via light microscopy (BX53; Olympus, Shinjuku, Japan) and transmission electron microscopy (Hitachi-HT7700, Tokyo, Japan). Gram staining was determined using a Gram Stain kit (QingDao Hopebio-Technology Co., Ltd., Qingdao, China) according to the manufacturer’s instructions. Gliding motility was performed as previously described [63]. Oxidase activity was tested with oxidase reagent (bioMérieux, Marcy-l’Étoile, France) and catalase activity was examined by bubble production in 3% (v/v) hydrogen peroxide. Nitrate reduction and the hydrolysis of agar, casein, DNA, starch and Tween 80 were carried out according to a previous description [64]. The temperature range for growth was assessed by incubating the strains at 4–40 °C (4, 10, 16, 20, 25, 28, 30, 33, 35, 37 and 40 °C). Growth at different pH values (5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 and 10) was performed in marine ZoBell broth (MB). For pH tolerance experiments, the following buffer solutions were used: MES (pH 5.5–6.0), PIPES (pH 6.5–7.0), HEPES (pH 7.5–8.0), Tricine (pH 8.5) and CAPSO (pH 9.0, 9.5 and 10.0) at a concentration of 20 mM. Growth at various NaCl concentrations was investigated in a modified MB made with 0.5% peptone, 0.1% yeast extract, 0.01% FePO4, artificial seawater (0.6% MgCl2, 0.32% Na2SO4, 0.18 % CaCl2, 0.06% KCl, 0.02% Na2CO3, traces of Na2SiO3, and NaF, w/v) and in the presence of 0–10% (w/v) NaCl (0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 %). Anaerobic growth (10% CO2, 5% H2 and 85% N2) and micro-aerobic growth (5% O2, 10% CO2 and 85% N2) were detected in an anaerobic chamber on MA plates with or without 0.25% (w/v) NaNO3 for 15 days at 30 °C. The antibiotic sensitivity of the strain was examined as recommended [65], using the antibiotic-impregnated discs (Hangzhou Microbial Reagent Co., Ltd., Hangzhou, China) in the following: norfloxacin (10 μg), gentamicin (10 μg), kanamycin (30 μg), cephalexin (30 μg), medilamycin (30 μg), chloramphenicol (30 μg), erythromycin (15 μg), clindamycin (2 μg), ampicillin (10 μg), penicillin (10 μg), ofloxacin (5 μg), vancomycin (30 μg), neomycin (30 μg), polymyxin B (300 IU), tetracycline (30 μg) and compound sulfamethoxazole (23.75/1.25 μg). Additional physiological and biochemical characteristics were tested using API 20NE, API 50CHB and API ZYM strips (bioMérieux), according to the manufacturer’s instructions, with the single modification of adjusting the salinity to 3% (w/v).

2.5. Chemotaxonomic Characterization

The cell biomass of strains for chemotaxonomic analysis was collected and freeze-dried after incubation in MB at 30 °C for 48 h. The respiratory isoprenoid quinones of strains 81s02T and 334s03T were extracted and analyzed by HPLC as described [66,67,68]. The polar lipids of novel isolates were extracted and detected using 2D thin-layer chromatography (TLC). Total lipid material was examined using molybdatophosphoric acid and specific functional groups were investigated using spray reagents specific for each, according to Tindall et al. [69]. The fatty acids were determined for the strains 81s02T and 334s03T, as well as for M. aquimarina SW-63T, M. aurea BC31-1-A7T, M. lutimaris SMK-108T and M. ruestringensis B1T, according to the standard protocol of the Microbial Identification System (MIDI, Sherlock Version 6.3). Fatty acids were methylated and analyzed using an Agilent 6890 N gas chromatography instrument (Santa Clara, CA, USA) and identified using the RTSBA6 database of the microbial identification system [70].

3. Results and Discussion

3.1. Phylogenetic and Genome Analysis

The almost full-length 16S rRNA gene sequences of strains 81s02T (1488 bp, OQ547168) and 334s03T (1488 bp, OQ547169) were determined and confirmed with the draft genome sequence. The strains shared 98.1% 16S rRNA gene sequence similarity to each other. Sequence similarity values calculated for the strains 81s02T and 334s03T indicated the greatest degree of similarity to M. lutimaris SMK-108T (98.7%) and M. aurea BC31-1-A7T (98.8%), respectively. Moreover, strain 81s02T exhibited 16S rRNA gene sequence similarities of 98.7, 98.6, 98.3, 98.2, 98.1, 98.1, 98.1 and 98.0% to the related strains M. aquimarina SW-63T, M. ruestringensis B1T, M. aurea BC31-1-A7T, M. abyssi W52T, M. brasiliensis K001T, M. oceanensis 40DY170T, M. oceani 501str8T and M. chongwuensis HICWT, respectively; and strain 334s03T exhibited 16S rRNA gene sequence similarities of 98.6, 98.5, 98.5, 98.5, 98.4, 98.4, 98.3 and 98.2% to the related strains M. abyssi W52T, M. aquimarina SW-63T, M. ruestringensis B1T, M. brasiliensis K001T, M. chongwuensis HICWT, M. oceani 501str8T, M. lutimaris SMK-108T and M. oceanensis 40DY170T, respectively. In addition, strains 81s02T and 334s03T showed less than 98.0% 16S rRNA gene sequence similarity to the other representative members within the genus Muricauda.
The draft genome sequence of strain 81s02T (JARFVA000000000) resulted in 14 contigs and yielded a genome of 4,030,812 bp in length after assembly. Contigs varied in length from 1908 to 1,216,753 bp, and the N50 value was 852,588. The draft genome sequence of strain 334s03T (JARFVB000000000) resulted in 44 contigs and yielded a genome of 4,292,830 bp in length after assembly. Contigs varied in length from 1164 to 968,529 bp, and the N50 value was 323,675. The sequencing depths of coverage were 347× and 294× for strains 81s02T and 334s03T, respectively. The genomic DNA G+C contents of strains 81s02T and 334s03T were 41.6 and 41.9 mol%, respectively, which were determined from the genome sequence. ANIb and ANIm values between new strains (81s02T and 334s03T) and reference strains ranged from 78.0% to 86.3%, which were significantly lower than the threshold value (95–96%) for the delineation of genomic species [71]. The dDDH values based on the draft genomes between new strains and reference strains ranged from 21.5% to 33.9%, which were far below cut-off values (70%) for species differentiation [57] (Table S1). Moreover, the ANIb, ANIm and dDDH values between strain 81s02T and 334s03T were 81.4–81.5, 85.5–85.6 and 25.4%, respectively. The overall topological structures of the phylogenetic and phylogenomic trees (Figure 1 and Figure 2) clearly showed that strains 81s02T and 334s03T fell within the clade comprising species of the genus Muricauda. The phylogenetic trees using MP and ML algorithms also showed essentially the similar topology (Figures S1 and S2).

3.2. Morphological, Physiological and Biochemical Characteristics

Cells of the strains 81s02T (0.4–0.6 × 1.0–3.0 μm) and 334s03T (0.3–0.6 × 0.8–2.5 μm) were rod shaped after 3 days’ growth in MB (Figure 3). The two novel strains, appeared to be sensitive to cephalexin (30 μg), medilamycin (30 μg), clindamycin (2 μg) and ofloxacin (5 μg), and thus showed a similar antibiotic sensitivity. However, strain 334s03T was sensitive to erythromycin (15 μg) and vancomycin (30 μg), while strain 81s02T was not. Growth was observed in microaerobic conditions for both strains, but no growth was observed under anaerobic conditions. Strains 81s02T and 334s03T also shared several common biochemical properties with the reference strains M. aquimarina SW-63T, M. aurea BC31-1-A7T, M. lutimaris SMK-108T and M. ruestringensis B1T. All abovementioned strains in this study were positive for the following: oxidase and catalase; the hydrolysis of aesculin and PNPG; acid production from d-glucose, d-fructose, d-mannose, MDM (methyl-α-d-mannopyranoside), MDG (methyl-α-d-glucopyranoside), amygdalin, arbutin, salicin, d-cellobiose, d-maltose, d-lactose, d-melibiose, sucrose, d-trehalose, d-melezitose, d-raffinose, starch, gentiobiose, d-turanose and potassium 5-ketogluconate (weak); and the activity of alkaline phosphatase, esterase lipase C4, esterase lipase C8, lipase (C14) (weak), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase and α-mannosidase. All abovementioned strains in this study were negative for the following: Gram-reaction; flexirubin-type pigment test; hydrolysis of agar and DNA; acid production from glycerol, d-ribose, l-xylose, d-adonitol, d-mannitol and d-sorbitol; and activity of α-fucosidase. However, the two novel strains differed markedly from strains M. aquimarina SW-63T, M. aurea BC31-1-A7T, M. lutimaris SMK-108T and M. ruestringensis B1T having a positive result for the growth at pH 9.5 and acid production from glycogen. In addition, strain 81s02T and 334s03T could be distinguished from each other by the tolerance of NaCl, acid production from N-acetylglucosamine, activity of N-acetyl-glucosaminidase and their ability to hydrolyze casein. The complete morphological, physiological and biochemical characteristics of strains 81s02T and 334s03T were shown in the species description and Table 1.

3.3. Chemotaxonomic Characteristics

For both strains, predominant lipoquinone was menaquinone-6 (MK-6), which was in accordance with the previous observations for species of the genus Muricauda [1,2,3,16,17,18,25,28,72,73,74]. The major fatty acids (>10%) of strains 81s02T and 334s03T were iso-C15:0, iso-C17:0 3-OH and iso-C15:1 G, followed by iso-C15:0 3-OH. The fatty acid compositions of both strains was similar to those of closely related type strains within Muricauda (Table 2). Nevertheless, there were several different characteristics between these strains. For instance, the contents of iso-C17:0 3-OH in 81s02T and 334s03T were significantly higher than those in M. aquimarina SW-63T, M. aurea BC31-1-A7T, M. lutimaris SMK-108T and M. ruestringensis B1T. However, C12:0, C15:0 3-OH and C17:0 3-OH were absent in strains 81s02T and 334s03T, while they were detected in the other type strains. The polar lipids of strain 81s02T comprised phosphatidylethanolamine (PE), one unidentified aminolipid (AL) and two unidentified lipids (L 1–2). Strain 334s03T comprised similar polar lipid components with strain J15B81-2T except for the absence of one unidentified aminolipid (AL) (Figure S3). Phosphatidylethanolamine, phospholipid and two unidentified lipids (L 1–2) were the common major polar lipids of novel isolates and their related type strains within the genus Muricauda. Meanwhile, the number of ALs identified in strains 81s02T and 334s03T was less than that in M. aquimarina SW-63T, M. lutimaris SMK-108T and M. ruestringensis B1T [24].
Table
Table 1. Differentiating characteristics of strains 81s02T and 334s03T from their closest phylogenetic relatives. Strains: (1) 81s02T; (2) 334s03T; (3) M. lutimaris SMK-108T; (4) M. aurea BC31-1-A7T; (5) M. aquimarina SW-63T; (6) M. ruestringensis B1T; (7) M. brasiliensis K001T [73]; (8) M. chongwuensis HICWT [72]; (9) M. oceanensis 40DY170T [75]; (10) M. oceani 501str8T [24]. Data for strains 1, 2, 3, 4, 5 and 6 were determined from this study unless indicated. +, positive; w, weakly positive; −, negative; ND, no data available.
Table 1. Differentiating characteristics of strains 81s02T and 334s03T from their closest phylogenetic relatives. Strains: (1) 81s02T; (2) 334s03T; (3) M. lutimaris SMK-108T; (4) M. aurea BC31-1-A7T; (5) M. aquimarina SW-63T; (6) M. ruestringensis B1T; (7) M. brasiliensis K001T [73]; (8) M. chongwuensis HICWT [72]; (9) M. oceanensis 40DY170T [75]; (10) M. oceani 501str8T [24]. Data for strains 1, 2, 3, 4, 5 and 6 were determined from this study unless indicated. +, positive; w, weakly positive; −, negative; ND, no data available.
Characteristic1234567 ♯8 ♯9 ♯10 ♯
Growth at:
 Temperature Range (°C)10–4010–4010–38 *10–40 *10–44 *8–40 *15–3715–4015–408–42
 Optimum temperature 3030–3530 *28–32 *30–37 *20–30 *28–3325–303525–30
 pH Range6.0–9.56.0–9.55.0–8.0 *6.0–8.5 *6.0–9.0 *6.0–9.0 *6.0–8.06.0–8.05.5–9.05.5–9.0
 Optimum pH7.0–7.57.57.0–8.0 *7.5 * 7.0 * 8.0 *ND76.5–7.07
 NaCl Range (%, w/v)0.5–10.00.5–9.01.0–10.0 *2.0–10.0 *0.5–9.0 *0.5–9.0 *0.5–9.00.5–8.00.5–10.00.5–10.0
Nitrate reduction−−−+w−−w−−
Indole production−−−+−−−ND−−
Hydrolysis of:
 Arginine−−−+−−NDND−+
 Casein+−+−++NDNDND−
 Gelatin−−−+−−−−−−
 Starch−−−−−−−NDw−
 Tween 80++−−+++ND+−
 Urease−−−+−−−ND−+
Acid production from:
 l-Arabinose−ww+−wNDND+ND
 d-Xylose−ww+w+NDND+ND
 d-Galactose+ww+wwNDND+ND
 l-Rhamnose−−−+−−NDNDwND
 N-Acetyl-glucosamine+−w++wNDNDNDND
 Inulin−−−−+wNDNDNDND
 Glycogen++w−−−NDNDNDND
Enzymic activities:
 Trypsin++−+++++++
 α-Chymotrypsin−−w++−+++−
 β-Glucuronidase−−+−−−−w+−
 N-Acetyl-glucosaminidase+−++++++++
DNA G+C content (mol%)41.641.940.142.143.441.441.641.442.442.8
* Data taken from the literature [1,2,18,74]. ♯ Data taken from the literature [24,72,73,75].
Table
Table 2. Fatty acid content of strains 81s02T and 334s03T in comparison with those of closely related strains. Strains: (1) 81s02T; (2) 334s03T; (3) M. aquimarina SW-63T; (4) M. aurea BC31-1-A7T; (5) M. lutimaris SMK-108T; (6) M. ruestringensis B1T. All data were obtained in this study. Fatty acids amounting to above 10% were in bold. Fatty acids amounting to less than 0.50% of the total in all strains were not included. TR, trace (<0.50 %); ND, not detected.
Table 2. Fatty acid content of strains 81s02T and 334s03T in comparison with those of closely related strains. Strains: (1) 81s02T; (2) 334s03T; (3) M. aquimarina SW-63T; (4) M. aurea BC31-1-A7T; (5) M. lutimaris SMK-108T; (6) M. ruestringensis B1T. All data were obtained in this study. Fatty acids amounting to above 10% were in bold. Fatty acids amounting to less than 0.50% of the total in all strains were not included. TR, trace (<0.50 %); ND, not detected.
Fatty Acid123456
Straight-chain:
 C12:0NDNDTRTR0.56 TR
 C16:01.95 2.07 0.74 0.93 0.54 1.25
 C18:00.74 0.64 TRNDTRTR
Branched:
 iso-C13:0TRTR0.79 0.84 1.07 0.81
 iso-C14:0TRND2.23 ND4.76 5.00
 iso-C15:039.0839.6038.2946.6136.8538.33
 iso-C15:1 G13.0715.0419.7610.6415.2315.18
 iso-C16:00.77 0.53 2.43 1.35 0.68 1.00
 iso-C17:00.77 TRTR1.00 TR0.74
 anteiso-C15:00.87 1.97 4.16 1.37 3.66 3.55
Unsaturated:
 C18:1 ω9c0.54 0.70 TRTRTRTR
Hydroxy:
 C15:0 3-OHNDND0.72 TR1.77 0.59
 C16:0 3-OH0.68 0.61 TRTR0.58 0.64
 C17:0 2-OHTRND0.83 TR1.34 0.98
 C17:0 3-OHNDNDTRTR1.45 0.59
 iso-C15:0 3-OH5.16 5.05 4.21 4.74 5.53 4.49
 iso-C16:0 3-OH1.53 0.85 3.98 1.51 2.53 1.92
 iso-C17:0 3-OH26.6827.7215.9522.4819.2118.97
Summed features: *
 20.59 0.59 TRTRTRTR
 32.05 2.00 1.28 1.72 0.96 1.34
 91.93 0.60 0.66 1.79 TR0.82
* Summed features are fatty acids that cannot be resolved reliably from another fatty acid using the chromatographic conditions chosen. The MIDI system groups these fatty acids together as one feature with a single percentage of the total. Summed feature 2 contained C14:0 3-OH and/or iso-C16:1 I; Summed feature 3 contained C16:1 ω7c and/or C16:1 ω6c; Summed feature 9 contained C16:0 10-methyl and/or iso-C17:1 ω9c.

3.4. Genome Attributes and Comparative Genome Analysis

The draft genome of strain 81s02T contained 3662 ORFs and 42 tRNAs, while strain 334s03T contained 3864 ORFs and 38 tRNAs. Pathway analyses on the KEGG website suggested that all eleven strains in this study within genus Muricauda had a complete glycolysis pathway (Embden–Meyerhof pathway, EMP) although the genes encoding some reaction steps were diverse (Figure 4). A complete pentose phosphate pathway (PPP) was also found in all eleven genomes, and it was more conserved compared to the EMP, where all genes encoding the PPP found in all strains were the same, aside from ripA, which was only found in strain M. oceani 501str8T. On the other hand, all strains were lacking the gene edd, encoding phosphogluconate dehydratase, indicating that the Entner–Doudoroff pathway might not present in Muricauda strains. Several Muricauda species contained the genes nasC and nirA, such as M. aurea, M. chongwuensis, M. brasiliensi, M. oceani and M. ruestringensis, while the other species only contained nirA but not nasC. Commonly, genes nasC and nirA were recognized to reduce nitrate to nitrite and reduce nitrite to ammonia, respectively [76]. Therefore, Muricauda species had a diverse nitrate reduction capability. Moreover, a complete assimilatory sulfate reduction pathway was found in M. ruestringensis B1T through the existence of genes cysC, cysD, cysN, cysJ and cysI. It was indicated that M. ruestringensis (the type species of genus Muricauda) had the capability of reducing sulfate to sulfite or hydrogen sulfide. However, other members in this study were lacking certain genes, showing an uncomplete sulfate reduction pathway. In addition, KEGG annotation showed that rhamnose containing glycans biosynthesis protein, polysaccharide biosynthesis/export protein, and lipopolysaccharide (dTDP-l-rhamnose) biosynthesis/assembly protein-related genes were found in all eleven Muricauda strains, and it has been suggested that it helps Muricauda survive in marine environments and assists them to endure extremes of temperature, salinity and nutrient availability. Metabolic features related to functional categories from RAST showed that genes associated with “virulence, disease and defense” existed in the genomes of Muricauda strains, which might be important for Muricauda to resist the toxic compounds in the environments (Figure S4). A large number of genes were involved in the class of “stress response”, which might provide these species the ability to adapt to special environments stresses, such as pressure, oxygen concentration, temperature, pH and salinity in marine ecosystem.
Carbohydrate-active enzymes (CAZymes) are involved in many metabolic pathways and in the biosynthesis and degradation of various biomolecules, such as bacterial exopolysaccharides, starch, cellulose and lignin [77]. Thus, genes putatively coding for carbon metabolism were analyzed among different species within Muricauda, including CAZymes, redox enzymes with auxiliary activities (AAs) and those with carbohydrate-binding modules (CBMs) (Figure S5). CAZyme families were classified into four major groups: glycoside hydrolases (GHs), glycosyltransferases (GTs), polysaccharide lyases (PLs) and carbohydrate esterases (CEs). Strains 81s02T and 334s03T harbored similar CAZyme-encoding genes compared to other species of Muricauda. These genes were mainly present in the groups of GHs and GTs. A number of GH3, GH13, GH109, GT2 and GT4 were identified in all genomes; however, GH43 was not observed in the genome of strain 81s02T, which was present in other ten strains. Moreover, PL7, PL9 and CBM62 were only observed in the genome of strain M. aurea BC31-1-A7T, while PL8 was only observed in the genome of strain M. chongwuensis HICWT, suggesting a different process for carbon metabolism in these strains. Therefore, a more expanded investigation for the carbon utilization of the genus Muricauda is required to gain insights into a complete process for the carbon metabolism in the future.

4. Conclusions

Taken together, the phylogenetic analysis, chemotaxonomic data and phenotypic results presented above suggested that 81s02T and 334s03T represent two novel species of the genus Muricauda, for which the names Muricauda okinawensis sp. nov. and Muricauda yonaguniensis sp. nov. are proposed, with 81s02T and 334s03T as type strains, respectively.

4.1. Description of Muricauda okinawensis sp. Nov.

Muricauda okinawensis (o.ki.na.wen’sis. N.L. masc./fem. adj. okinawensis, pertaining to Okinawa Trough, where the type strain was isolated.)
Cells are Gram-staining negative, facultatively anaerobic rods (0.4–0.6 × 1.0–3.0 μm) non-flagellated and non-gliding. Colonies on MA are circular, smooth, yellow-pigmented and about 1.5 mm in diameter after 2 days of growth at 30 °C. Growth occurs at 10–40 °C and in the presence of 0.5–10.0% (w/v) NaCl, with the optimum growth at 30 °C and with 2% (w/v) NaCl. The optimum pH is 7.0–7.5 and no growth occurs below pH 6.0 or above pH 9.5. A flexirubin-type pigment is not produced. It is positive for catalase and oxidase. Negative for nitrate reduction, indole and H2S production. It is positive for the hydrolysis of aesculin, casein, PNPG and Tween 80, but negative for the hydrolysis of agar, arginine, DNA, gelatin, starch and urea. Acid is produced from d-glucose, d-fructose, d-mannose, MDM, MDG, amygdalin, arbutin, salicin, d-cellobiose, d-maltose, d-lactose, d-melibiose, sucrose, d-trehalose, d-melezitose, d-raffinose, starch, gentiobiose, d-turanose, potassium 5-ketogluconate (weak), d-galactose, N-acetyl-glucosamine and glycogen. Alkaline phosphatase, esterase lipase C4, esterase lipase C8, lipase (C 14) (weak), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-glucosaminidase and α-mannosidase are present, while α-chymotrypsin, β-glucuronidase and α-fucosidase are absent. The major fatty acids are iso-C15:0, iso-C17:0 3-OH and iso-C15:1 G. Menaquinone-6 (MK-6) is the predominant respiratory quinone. The major polar lipids are phosphatidylethanolamine and two unidentified lipids. The DNA G+C content of the type strain is 41.6 mol%.
The type strain is 81s02T (=KCTC 92889T = MCCC 1K08502T) and was isolated from the sediment core near the hydrothermal fields of the southern Okinawa Trough. The GenBank accession numbers for the 16S rRNA gene and the draft whole genome data of the strain 81s02T are OQ547168 and JARFVA000000000, respectively.

4.2. Description of Muricauda yonaguniensis sp. Nov.

Muricauda yonaguniensis (Yo.na.gu.ni.en ‘sis N.L. masc./fem. adj. Yonaguniensis, pertaining to the Yonaguni Knoll IV hydrothermal field, where the type strain was isolated.)
Cells are Gram-staining negative, facultatively anaerobic rods (0.3–0.6 × 0.8–2.5 μm), and are non-flagellated and non-gliding. Colonies on MA are circular, smooth, yellow-pigmented and about 1.5 mm in diameter after 2 days of growth at 30 °C. Growth occurs at 10–40 °C and in the presence of 0.5–9.0% (w/v) NaCl, with the optimum growth at 30–35 °C and with 2% (w/v) NaCl. The optimum pH is 7.5 and no growth occurs below pH 6.0 or above pH 9.5. A flexirubin-type pigment is not produced. It is positive for catalase and oxidase. It is negative for nitrate reduction, indole and H2S production. It is positive for the hydrolysis of aesculin, PNPG and Tween 80, but negative for the hydrolysis of agar, arginine, casein, DNA, gelatin, starch and urea. Acid is produced from d-glucose, d-fructose, d-mannose, MDM, MDG, amygdalin, arbutin, salicin, d-cellobiose, d-maltose, d-lactose, d-melibiose, sucrose, d-trehalose, d-melezitose, d-raffinose, starch, gentiobiose, d-turanose, potassium 5-ketogluconate (weak), d-galactose, l-arabinose (weak), d-xylose (weak) and glycogen. Alkaline phosphatase, esterase lipase C4, esterase lipase C8, lipase (C 14) (weak), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase and α-mannosidase are present, while α-chymotrypsin, β-glucuronidase, N-acetyl-glucosaminidase and α-fucosidase are absent. The major fatty acids are iso-C15:0, iso-C17:0 3-OH and iso-C15:1 G. Menaquinone-6 (MK-6) is the predominant respiratory quinone. The major polar lipids are phosphatidylethanolamine and two unidentified lipids. The DNA G+C content of the type strain is 41.9 mol%.
The type strain is 334s03T (=KCTC 92890T = MCCC 1K08503T) and was isolated from the sediment core near the hydrothermal fields of the southern Okinawa Trough. The GenBank accession numbers for the 16S rRNA gene and the draft whole genome data of strain 334s03T are OQ547169 and JARFVB000000000, respectively.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms11061580/s1, Figure S1: Phylogenetic tree, based on the 16S rRNA gene sequences using the maximum likelihood algorithm showing the position of strains 81s02T and 334s03T. GenBank accession numbers used are given in the parentheses. Bootstrap values higher than 50% are indicated at branch nodes. Formosa maritima 1494T was used as an outgroup. Bar, 0.02 substitutions per nucleotide position; Figure S2: Phylogenetic tree, based on the 16S rRNA gene sequences using the maximum parsimony algorithm showing the position of strains 81s02T and 334s03T. GenBank accession numbers used are given in the parentheses. Bootstrap values higher than 50% are indicated at branch nodes; Figure S3: Two-dimensional thin-layer chromatogram of polar lipids; Figure S4: Metabolic features related to functional categories of 11 Muricauda strains. The encoding gene involved in the categories of “Photosynthesis”, “Iron acquisition and metabolism” and “Motility and Chemotaxis” was not annotated; Figure S5: Genes putatively coding for carbon metabolism among different Muricauda species based on the CAZy database. Table S1: ANIb, ANIm and dDDH values between pairs of type strains of Muricauda species.

Author Contributions

Strain 8102T and 334s03T were isolated by X.D. and W.C. Material preparation, data collection and analysis were performed by W.C., X.D. and M.J. The samples was collected and provided by Z.Z. The original draft was written by W.C. The writing, review and editing for the manuscript was performed by M.J., Z.Z. and F.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Natural Science Foundation of Shandong Province, China (No. ZR2020MD088); the National Natural Science Foundation of China (No. 41976202); the Open Fund of the Key Laboratory of Marine Geology and Environment, Chinese Academy of Sciences (Nos. MGE2022KG4 and MGE2022KG6); the National Basic Research Program of China (No. 2013CB429700); and the Taishan Scholars Program of Shandong Province (No. ts201511061).

Data Availability Statement

The datasets used during the current study are available from the corresponding author on reasonable request.

Acknowledgments

Special thanks to Bowen Zhu (Key Laboratory of Marine Geology and Environment, Institute of Oceanology, Chinese Academy of Sciences) for the sample provision. We thank Yuanyuan Sun (Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences) for assistance with TEM operations. We also thank the anonymous reviewers and editors for their insightful suggestions and careful reading.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

MES2-N-Morpholino ethanesulfonic acid hydrate
PIPESPiperazine -1, 4-bis (2-ethanesulfonic acid)
HEPES2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic acid
CAPSOCapso sodium salt
PNPG4-Nitrophenyl β-d-glucopyranoside

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Microorganisms 11 01580 g001 550
Figure 1. Phylogenetic tree, based on the 16S rRNA gene sequences using the neighbor-joining algorithm showing the position of strains 81s02T and 334s03T. GenBank accession numbers used are given in the parentheses. Open circles indicated that the branches were recovered in maximum likelihood or maximum parsimony analyses. Filled circles showed branches that were recovered in maximum likelihood and maximum parsimony analyses. Bootstrap values higher than 50% were indicated at branch nodes. Formosa maritima 1494T was used as an outgroup. Bar, 0.02 substitutions per nucleotide position.
Figure 1. Phylogenetic tree, based on the 16S rRNA gene sequences using the neighbor-joining algorithm showing the position of strains 81s02T and 334s03T. GenBank accession numbers used are given in the parentheses. Open circles indicated that the branches were recovered in maximum likelihood or maximum parsimony analyses. Filled circles showed branches that were recovered in maximum likelihood and maximum parsimony analyses. Bootstrap values higher than 50% were indicated at branch nodes. Formosa maritima 1494T was used as an outgroup. Bar, 0.02 substitutions per nucleotide position.
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Figure 2. Phylogenomic tree of new strains (81s02T and 334s03T) and other available type strains of the most closely related species based on 120 conserved concatenated proteins (Bac120 sets) from genomes sequences from the NCBI GenBank database. GenBank accession numbers used were given in the parentheses. Formosa maritima 1494T was used as an outgroup. Bar, 0.1 substitutions per amino acid position.
Figure 2. Phylogenomic tree of new strains (81s02T and 334s03T) and other available type strains of the most closely related species based on 120 conserved concatenated proteins (Bac120 sets) from genomes sequences from the NCBI GenBank database. GenBank accession numbers used were given in the parentheses. Formosa maritima 1494T was used as an outgroup. Bar, 0.1 substitutions per amino acid position.
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Figure 3. Cell micrograph of strains 81s02T and 334s03T grown in marine ZoBell broth for 3 days taken using transmission electron microscopy (Hitachi-HT7700). (a) 81s02T; (b) 334s03T.
Figure 3. Cell micrograph of strains 81s02T and 334s03T grown in marine ZoBell broth for 3 days taken using transmission electron microscopy (Hitachi-HT7700). (a) 81s02T; (b) 334s03T.
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Figure 4. Presence and absence of metabolic pathway genes in eleven Muricauda strains. The hollow circles indicate no gene-related functions found in the genomes. The solid circles represent genes found in the genomes.
Figure 4. Presence and absence of metabolic pathway genes in eleven Muricauda strains. The hollow circles indicate no gene-related functions found in the genomes. The solid circles represent genes found in the genomes.
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Cao, W.; Deng, X.; Jiang, M.; Zeng, Z.; Chang, F. Muricauda okinawensis sp. Nov. and Muricauda yonaguniensis sp. Nov., Two Marine Bacteria Isolated from the Sediment Core near Hydrothermal Fields of Southern Okinawa Trough. Microorganisms 2023, 11, 1580. https://doi.org/10.3390/microorganisms11061580

AMA Style

Cao W, Deng X, Jiang M, Zeng Z, Chang F. Muricauda okinawensis sp. Nov. and Muricauda yonaguniensis sp. Nov., Two Marine Bacteria Isolated from the Sediment Core near Hydrothermal Fields of Southern Okinawa Trough. Microorganisms. 2023; 11(6):1580. https://doi.org/10.3390/microorganisms11061580

Chicago/Turabian Style

Cao, Wenrui, Xingyu Deng, Mingyu Jiang, Zhigang Zeng, and Fengming Chang. 2023. "Muricauda okinawensis sp. Nov. and Muricauda yonaguniensis sp. Nov., Two Marine Bacteria Isolated from the Sediment Core near Hydrothermal Fields of Southern Okinawa Trough" Microorganisms 11, no. 6: 1580. https://doi.org/10.3390/microorganisms11061580

APA Style

Cao, W., Deng, X., Jiang, M., Zeng, Z., & Chang, F. (2023). Muricauda okinawensis sp. Nov. and Muricauda yonaguniensis sp. Nov., Two Marine Bacteria Isolated from the Sediment Core near Hydrothermal Fields of Southern Okinawa Trough. Microorganisms, 11(6), 1580. https://doi.org/10.3390/microorganisms11061580

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