Muricauda chongwuensis sp. nov., isolated from coastal seawater of China

In the course of screening for bacterial predators, a Gram-stain-negative, non-flagellated, gliding, long rod-shaped, and yellow-pigmented bacterium, designated strain HICWT, was isolated from coastal seawater of China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain HICWT represented a member of the genus Muricauda and showed the highest sequence similarity to M. aquimarina JCM11811T (98.8%) and M. ruestringensis DSM13258T (98.1%). The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between strain HICWT and M. aquimarina JCM11811T were 79.2% and 34.1%, respectively. NaCl was required for growth. Optimum growth occurred at 25–30 °C, 2.0–3.0% (w/v) NaCl with pH 7.0. Strain HICWT showed some similar characteristics to the nonobligate bacterial predators, and the cells can attach to the prey cells. Strain HICWT contained MK-6 as the predominant respiratory quinone and had iso-C15:0, iso-C15:1 G, and iso-C17:0 3-OH as the major cellular fatty acids. The polar lipids contained phosphatidylethanolamine (PE), three unidentified phospholipids (PL1–PL3), one unidentified amino lipids (AL), and three unidentified polar lipids (L1–L3). The genome size of strain HICWT was approximately 3.8 Mbp, with a G + C content of 41.4%. Based on the polyphasic evidence, strain HICWT is proposed as representing a new species of the genus Muricauda, for which the name Muricauda chongwuensis sp. nov. is proposed. The type strain is HICWT (= JCM 33643 T = MCCC 1K03769T).


Introduction
According to the LPSN (www. bacte rio. net/ index. html), the family Flavobacteriaceae comprises 145 genera, including genus Muricauda, which was proposed by Bruns et al. (2001), and subsequently emended by Yoon et al. (2005) and Hwang et al. (2009). Members of the genus Muricauda share the characteristics of being Gram-stain-negative, non-motile, strictly or facultatively aerobic, yellow-pigmented rod, and having DNA G + C contents of 41.0-55.0 mol%. At the time of writing, there are 28 valid species in the genus Muricauda listed in the LPSN (https:// lpsn. dsmz. de/ genus/ muric auda). These Muricauda species were isolated from various saline Communicated by Erko Stackebrandt.
* Ming-Xia Chen chenmx1257@163.com; chenmx1257@hqu.edu.cn environments, such as intertidal/tidal flat, salt lake, seawater and sediment, sponge, shrimp gill, rhizosphere of marine macroalga and phycosphere of dinoflagellate (Yoon et al. 2005;Yoon et al. 2008;Yoon and Oh 2012;Bae et al. 2007;Dang et al. 2019;Guo et al. 2020;Kim et al. 2020;Park 2019;Liu et al. 2020;Zhang et al. 2020;Chen et al. 2021;Zhu et al. 2021). Predatory bacteria can be any bacteria that kill or destroy other microbes and consume them as a nutritional resource (Pérez et al. 2016). Most described predatory bacteria except members of Bdellovibrio-and-like organisms (BALOs) (Williams and Chen 2020) are nonobligate predators, such as Ensifer adhaerens (Germida and Casida 1983), Agromyces ramosus and Lysobacter (Jurkevitch and Davidov 2006;Svercel et al. 2011), Pseudobacteriovorax antillogorgiicola (Mccauley et al. 2015), Bradymonas sediminis (wang et al. 2015;Mu et al. 2020), Wenzhouxiangella Strain AB-CW3 (Sorokin et al. 2020). In the course of screening for bacterial predators distributed in the coastal waters of China, a yellow-pigmented strain, designated HICW T , was isolated. Strain HICW T showed some similar characteristics to nonobligate bacterial predators, and the results of 16S rRNA gene sequence comparisons indicated that it was phylogenetically related to the genus Muricauda in the family Flavobacteriaceae. The present study determined the taxonomic status of strain HICW T using a polyphasic approach.

Strain and culture condition
The sample was collected from coastal waters near the town Chongwu in Southeast China (118.545685° E, 24.53178° N). The sample was brought to the laboratory and stored at 4 °C (refrigerator) for 2 days before being processed. Strain HICW T was isolated and purified over five times using the seawater double-layer agar plating method as previously described with Vibrio alginolyticus LF TCBS 15 (= MCCC 1K03520) as the prey bacterium (Schoeffield and Williams 1990;Ye et al. 2019). Cells from the plaque were examined by light microscopy (CX22RFS1, OLYMPUS) with 1% (w/v) crystal violet staining. A single-plaque (with slender rod cells of strain HICW T and a few residual prey cells) on the double-layer plate was transferred into a 20 ml tubetype bottle with 2 ml 1/40 concentration of marine broth 2216E (MB, peptone 5 g, yeast extract 1 g, seawater 1 L, pH 7.2-7.6) and incubated at 28 °C with 200 r.p.m for 2-3 days. Small amounts of cells (each around 1 × 10 5-6 cell ml -1 ) of strain HICW T and prey strain LF TCBS 15 were detected in 1/40 (v/v) MB culture. The axenic independent strain HICW T was purified from the 1/40 (v/v) MB co-culture by the standard dilution plating on marine agar 2216E (MA, pH 7.2-7.6) after incubation at 28 °C for 6-7 days. A yellow colony different from the prey strain was picked, checked by light microscopy (CX22RFS1, OLYMPUS) with 1% (w/v) crystal violet staining, then purified by streaking three times on MA. The strain was maintained in MB at 28 °C for 24 h and preserved in MB supplemented with 20% (v/v) glycerol at − 20 °C and − 80 °C. For long-term storage, the cultures of strain HICW T were lyophilized in 10% (w/v) skim milk and then deposited at the Marine Culture Collection of China (MCCC) and Japan Collection of Microorganisms (JCM). M. aquimarina JCM11811 T and M. ruestringensis DSM13258 T were obtained from the Marine Culture Collection of China and cultivated under identical conditions.

Phenotypic and biochemical characterization
Cell morphology was observed by light microscopy (CX22RFS1, OLYMPUS) and transmission electron microscopy (TEM, HT7800, Hitachi). For negative stains, cells from 24 h cultures on MA were resuspended with 0.1 mol l −1 phosphate buffer (pH7.4), then a 400-mesh grid was inverted over a drop of cell suspensions for 1 min. The grid was then washed on two drops of water, and the cells were stained with 2.0% (w/v) uranyl acetate for 10 s.
For predatory characteristic detection, strain HICW T was cultivated either in double-layer agar plates or seawater with washed prey cells (around 1 × 10 9 cell ml -1 ) (Ye et al. 2019). Light microscopy (CX22RFS1, OLYMPUS) and transmission electron microscopy (TEM, HT7800, Hitachi) were used to assess the cell-to-cell contact with attachment to the prey. For negative stains, a 400-mesh grid was inverted over a drop of 24 h co-cultures seawater, washed on one drop of water, and the cells were stained as mentioned above.

Chemotaxonomic characterization
For analysis of the cell fatty acids, bacteria were cultured in MB at 28 °C for 24 h, and the harvested cells were saponified, methylated, and extracted using the standard MIDI (Sherlock Microbial Identification System, version 6.0B) protocol. The whole-cell fatty acid pattern was then analyzed by gas chromatography (model 6850, Agilent Technologies) and identified using the TSBA6.0 database of the Microbial Identification System (Athalye et al. 1985;Sasser 1990). Polar lipids were extracted and examined using twodimensional thin-layer chromatography according to Kates (1972). Isoprenoid quinones were extracted from freezedried cells with chloroform/methanol (2:1, v/v) and analyzed by reversed-phase HPLC (Collins et al. 1984).

Phylogenetic and genomic analyses
The genomic DNA of strain HICW T was extracted using a Rapid Bacterial Genomic DNA Isolation Kit (B518225, Shanghai Sangon Biological Engineering Technology & Services Co., Ltd). The whole genome of strain HICW T was sequenced by the Guangdong Magigene Biotechnology Co., Ltd., using the Solexa paired-end (150 bp library) sequencing technology protocol. SPAdes software (http:// cab. spbu. ru/ softw are/ spades/) was used to do genome assembly with multiple-Kmer parameters (Bankevich et al. 2012). The draft genome data of HICW T has been deposited in GenBank with the accession number WYET00000000. The G + C contents of the genomic DNA were calculated from the sequenced genome (https:// www. ezbio cloud. net/ tools/ ani). Open Reading Frames (ORFs) were predicted using Prodigal v2.6.3 (Hyatt et al. 2010), and the predicted protein-coding sequences (CDS) were searched against the GenBank, Clusters of Orthologous Groups (COGs), and KEGG databases to analyze gene functions and metabolic pathways.
The partial 16S rRNA gene (around 1400 bp) was amplified from the chromosomal DNA and obtained from plaques of the strain HICW T on the double-layer agar plate with V. alginolyticus LF TCBS 15 as the prey cells. The universal bacterial primers 27F and 1492R (Delong 1992) were used, and the purified PCR product was sequenced by Xiamen Bioray Biotechnology Co., Ltd. The complete 16S rRNA gene sequence of the strain HICW T was obtained from its draft genome sequence. The 16S rRNA gene sequence analyses were carried out with the online tool EzBioCloud (http:// eztax on-e. ezbio cloud. net) (Kim et al. 2012). 16S rRNA gene sequences of related taxa were selected from the GenBank database. Phylogenetic trees were reconstructed using the MEGA software package version 7.0 (Kumar et al. 2016) with distance options according to the default parameter model and clustering with the neighbor-joining (NJ), maximum-likelihood (ML), and maximum-parsimony (MP) methods, supported using bootstrap values with 1000 replications.
The whole-genome average nucleotide identity (gANI) was calculated using the algorithm as described by Yoon et al. (2017) with the web service of EzBioCloud (https:// www. ezbio cloud. net/ tools/ ani). The digital DNA-DNA hybridizations (dDDH) were determined online at http:// ggdc. dsmz. de/ ggdc. php# using the Genome-to-Genome Distance Calculation (GGDC) version 2.1 (Meier-Kolthoff et al. 2013). Genomic data of related species were downloaded from the GenBank database.

Phenotypic and biochemical characteristics
Cells of strain HICW T were Gram-stain-negative, slender rods without any flagella, 1.8-3.7 μm in length, 0.3-0.4 μm in width after culturing on MA for 24 h at 28 °C (Table 1, Fig. S1). Outer membrane vesicles (OMVs), the spherical buds of the outer membrane, detached from the bacterial cell surface were detected (Fig. 1). The strain formed small circular yellow colonies on MA for 72 h at 28 °C (Fig. S2a). Colony spreading was observed on MA with 1.0% agar (w/v) (Fig. S2b), and cells glided slowly at the bottom surface of the coverslip. Flexirubin-type pigments were not detected, but carotenoid pigments with maximal absorption at 454 nm and 480 nm were present (Fig. S3). The carotenoid pigments were yellow and non-diffusible. Nitrate reduction, gelatin hydrolysis, assimilation profile of several substrates, enzymatic activities, and antibiotic susceptibility profiles were the physiological properties differentiating among strain HICW T and closely related species of the genus Muricauda (Table 1, Table S1-S3). Other physiological and biochemical characteristics of strain HICW T are given in Table 1, Fig.  S4, and the species description.
Plaques were formed on lawns of V. alginolyticus LF TCBS 15 when strain HICW T was isolated, purified, and cultivated using double-layer agar plates in the first 6 months. Arc-shaped concaves on the plate became visible after a 24 h incubation at 28 °C, then they extended slowly and turned to be clear sunken plaques (Fig. S5). When the plaques were picked and incubated in seawater with prey cells (around 1 × 10 9 cell ml -1 ) at 28 °C for 48-120 h, strain HICW T showed a poor cell growth, the turbidity of the seawater co-cultured system did not decrease obviously, and the cells of strain HICW T could not be separated from prey cells using the centrifugation or membrane filtration. Subsequently, a 1/40 concentration of MB was used to incubate the plaques, in which the growth of strain HICW T and prey was small and in the same order of magnitude. The method of standard dilution plating on MA was tried to purify the axenic independent The tests for oxidase activities, the API 20NE, and the API ZYM strip were performed on strain HICW T and the related type strains in this study. Genome sequences of the related type strains were taken from GenBank and analyzed in this study. Other data for the related type strains were taken from their original description (Bruns et al. 2001;Yoon et al. 2005).   . 1 Transmission electron microscopy image of strain HICW T cells of strain HICW T from the 1/40 MB co-cultured system. Yellow colonies of strain HICW T different from the prey strain were detected on 10 -3 diluted plates. Accordingly, the number of strain HICW T in the 1/40 MB cocultured system was estimated at 10 5 CFU ml -1 , which was consistent with the results of microscope counting (10 5-6 cell ml -1 ). These results also indicated that strain HICW T did not belong to an obligate predator. However, after purification, the axenic independent cells of strain HICW T appeared to lose plaque-forming activity against V. alginolyticus LF TCBS 15. The same phenomenon showed on the cells from the early co-cultured systems (preserved with 20% (v/v) glycerol at − 20 °C or − 80 °C). A similar property was reported on Pseudobacteriovorax antillogorgiicola RKEM611 to lose predatory activity after subsequent transfers on solid media (Mccauley et al. 2015). In a seawater co-cultured system, strain HICW T could attach to the prey cells, one cell attached to one or more prey (Figs. 2, and S6). Empty prey cells adjacent to the cell of strain HICW T were detected (Fig. 2d). Although strain HICW T showed some similar characteristics to the nonobligate bacterial predators, the predation activity was weak in the liquid co-culture system. The predatory mechanism of strain HICW T has not yet been elucidated. More conclusive pieces of evidence are needed to prove strain HICW T as a bacterial predator. Here, we defined it as a potential predator or quasi-predator.

Phylogenetic and genomic analyses
Genome features of strain HICW T are summarized in length of 3,434,949 bp were predicted, which account for 90.9% of the genome, and 37 tRNA and five rRNA (one 23S rRNA, one 16S rRNA, and three 5S rRNA) genes were identified. In all, 2896 CDSs were assigned to COG families, and 1714 CDSs were included in 202 pathways. The partial 16S rRNA gene sequence (800 bp) from the sunken plaques and genomic DNA of strain HICW T showed most closely related to the genus Muricauda (around 83.0% and 97.6% similarity, respectively). The complete 16S rRNA gene sequence from the genome sequence of strain HICW T was 1514 bp in length. Comparisons of the 16S rRNA gene sequence with the corresponding ones in the EzBioCloud databases showed that strain HICW T shared the highest sequence similarity with the 16S rRNA gene of M. aquimarina JCM11811 T (98.8%) and M. ruestringensis DSM13258 T (98.1%). In the neighbor-joining tree based on 16S rRNA gene sequences of strain HICW T and related-type strains, the new isolate belonged to the family Flavobacteriaceae, fell into the same cluster with the members of the genus Muricauda and was most closely related to M. aquimarina JCM11811 T (Fig. 3). The maximum-parsimony and maximum-likelihood trees showed essentially the same topology (Figs. S8 and S9).
The ANI value for comparisons between strain HICW T and M. aquimarina JCM11811 T and M. ruestringensis DSM13258 T were79.2% and 80.6% (Table 1), respectively, which were lower than the threshold of 94-96% for bacterial species delineation (Kim et al. 2014;Richter and Rosselló-Móra 2009). The dDDH relatedness for strain HICW T with M. aquimarina JCM11811 T and M. ruestringensis DSM13258 T were 34.1% and 34.5% (Table 1), respectively, which were also clearly below the 70% threshold DDH value generally accepted for the delineation of species (Meier-Kolthoff et al. 2013).

Taxonomic conclusion
Based on the results of phenotypic, biochemical, chemotaxonomic, phylogenetic, and genomic analyses, it is clear that strain HICW T is genetically distinct from other strains of the genus Muricauda and represents a new species of the genus Muricauda, for which the name Muricauda chongwuensis sp. nov. is proposed.

Supplementary Information
The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s00203-021-02591-1. Data availability The GenBank/EMBL/DDBJ accession numbers for the draft genome sequence and the 16S rRNA gene sequence of Muricauda chongwuensis HICW T are WYET00000000 and MK920190, respectively.
Code availability Not applicable.

Conflict of interest
The authors declare that they have no conflict of interest.
Ethical statement This article does not contain any studies with human participants or animals performed by any of the authors.