Maricaulis alexandrii sp. nov., a novel dimorphic prosthecate and active bioocculants-bearing bacterium isolated from phycosphere microbiota of laboratory cultured highly-toxic Alexandrium catenella LZT09

An aerobic, Gram-stain-negative, straight or curved rods, prosthecate bacterium designated as LZ-16-1 T was isolated from phycosphere microbiota of highly-toxic and laboratory cultured dinoagellate Alexandrium catenella LZT09. This new isolate produces active bioocculanting exopolysaccharides (EPS). Cells were dimorphic with non-motile prostheca, or non-stalked and motile by a single polar agellum. Growth occurred at 10-40 °C, pH 5–9 and 1–8 % (w/v) NaCl, with optimum growth at 25 °C, pH 7–8 and 2-4 % (w/v) NaCl, respectively. Phylogenetic analysis based on 16S rRNA indicated that strain LZ-16-1 T was aliated to the genus Maricaulis, and closely related to Maricaulis parjimensis MCS 25 T (99.48%) and M. virginensis VC-5 T (99.04%),. However, based on genome sequencing and phylogenomic calculations, the average nucleotide identity (ANI) and digtal DNA-DNA genome hybridization (dDDH) values between the two strains were only 85.0 and 20.9%, respectively. Strain LZ-16-1 T owned Q-10 as predominant isoprenoid quinone; summed feature 8, C 16:0 , C 17:0 , C 18:0 , C 18:1 ω9c and summed feature 9 as dominant fatty acids; and sulfoquinovosyl diacylglycerol, glycolipids and unidentied phospholipid as major polar lipids. The genomic DNA G+C content is 63.6 mol%. Physiological and chemotaxonomic characterization further conrmed the distinctiveness of strain LZ-16-1 T from other Maricaulis members. Thus, strain LZ-16-1 T represents a novel species of the genus Maricaulis, for which the name Maricaulis


Introduction
The genus of Maricaulis was originally derived from the genus Caulobacter within the family Hyphomonadaceae of the order Rhodobacterales, and composed of dimorphic and prosthecate bacteria (Abraham et al. 1999;2002). Recently, the genus Maricaulis has been transferred to the newly-proposed family Maricaulaceae in the order Maricaulales by Kevbrin et al (2021). At the time of writing, the genus of Maricaulis contains ve validly named species all isolated from seawater (https://lpsn.dsmz.de/genus/ maricaulis) (Abraham et al. 2002). The division mode representing unique regulation feature of prokaryotic reproduction cycle of the dimorphic prosthecate bacteria comes from their evolution to oligotrophic habitats by minimizing competition under limited resources circumstances. Maricaulis are ubiquitous in aquatic environments with vital ecologically importance responsible for considerable mineralization of dissolved organic material (DOM) in water especially when the nutrient concentrations are low (Jannasch and Jones, 1960).
Marine phycosphere as the boundary of phytoplankton holobionts harbors dynamic host-microbe interactions and play crucial roles in aquatic ecosystems (Amin et al. 2012;Seymour et al. 2017;Zhang et al. 2020). To unveil the nature of those cross-kingdom associations, we initiated the Phycosphere Microbiome Project (PMP) to convey the microbial structures of phycosphere microbiota (PM) of diverse harmful algal blooms (HAB) dino agellates (Duan et al. 2020;Yang et al. 2018aYang et al. , 2018bYang et al. , 2020aYang et al. , 2020bYang et al. 2020;Zhang et al. 2015bZhang et al. , 2020Zhou et al. 2021). During the subsequent culture-dependent investigation which is a crucial prerequisite for the interactions study, a novel cultivable dimorphic and prosthecate bacterium designated as LZ-16-1 T was isolated from one dominant PM of Alexandrium catenella LZT09 (Fig. S1), which is a routinely laboratory cultured HAB dino agellate that produces high levels of paralytic shell sh poisoning toxins (PSTs). This new isolate produces active bio occulanting exopolysaccharides (EPS) (Fig. S2) demonstrating potential environmental and biological applications (Mu et al. 2019). In this study, we described the polyphasic characterization of strain LZ-16-1 T to represent a novel species of the genus Maricaulis.

Materials And Methods
Bacterial strains and culture conditions Strain LZ-16-1 T was isolated from Alexandrium catenella LZT09 by spreading the algal culture on marine agar (MA, Difco) plates according to our protocol described previously (Yang et al. 2018a(Yang et al. , 2018b(Yang et al. , 2020. The strain was puri ed and maintained on MA and preserved as a glycerol suspension (20%, v/v)

Phenotypic characterizations
Phenotypic characterizations were performed as described previously by Macián et al. (2005). Cell morphology of an exponentially growing culture of strain LZ-16-1 T was observed by transmission electron microscopy (JEM-1200; JEOL, Tokyo, Japan) using cultures grown on MA at 25 °C for 3 days. Motility was examined microscopically under the phase-contrast mode by the hanging drop technique. Gramstaining test was determined as described previously (Beveridge et al. 2007). Growth at different temperatures from 5 to 50 °C at 5 °C increments, pH range from 4.0 to 12.0 in increments of 0.5 pH unit) were determined as reported previously (Zhou et al. 2021;Yang et al. 2018b;Zhang et al. 2020). NaCl tolerance was determined in MB medium supplemented with 0-12% NaCl (w/v) at 25 °C for two weeks on a rotary shaker. Utilization of carbon sources and enzyme activities were tested using the API 20E and API ZYM (bioMérieux, Marcy-l'Étoile, France) strips following the manufacturer's instructions.

16S rRNA gene phylogenetic analysis
The extraction of genomic DNA, PCR ampli cation of the 16S rRNA gene using the universal primer of 27F/1492R, and sequencing of the PCR product were performed as described previously (Yang et al. 2018a(Yang et al. , 2018b. The 16S rRNA gene sequence similarities were compared with the sequences of the type strains available from GenBank using the BLAST and Ez-Taxon server (https://www.ezbiocloud.net).
Phylogenetic analysis of the 16S rRNA gene sequences of strain LZ-16-1 T (GenBank accession MK100326) and the reference type strains were performed using MEGA sofaware version 7.0 (Kumar et al. 2016). Phylogenetic trees were reconstructed by neighbour-joining (NJ), maximum likelihood (ML) and maximum parsimony (MP) algorithms using bootstrap analysis based on 1000 replications (Yang et al. 2018a(Yang et al. , 2018b. The Kimura's two-parameter model is used to generated the distance matrix (Kimura 1980).

Chemotaxonomic characterization
For the extraction of the quinones and polar lipids, the strains were harvested at stationary growth phase after growth in marine broth (MB) for 2 days at 25 °C. Polar lipids were extracted and separated by twodimensional TLC on silica gel 60 F254 plates (Merck 5554) and then analyzed by the method of by Minnikin et al. (1984). The respiratory quinone was isolated, puri ed and identi ed according to the method (Hiraishi et al. 1996). Cellular fatty acids were extracted, methylated and analyzed following the instructions of the Microbial Identi cation System (MIDI) (Sherlock version 6.1; MIDI database TSBA6).

Morphological, physiological and biochemical analyses
The morphological observation of strain LZ-16-1 T were performed on MA plates and formed circular, smooth and white colonies with 1.5-2.5 mm in diameter after 48h incubation at 25 °C. Cells of strain LZ-16-1 T were Gram-stain-negative, aerobic, oxidase-negative but catalase-positive, straight or curved rodshaped, Cells usually possess a prostheca ca. 0.15 μm in diameter and of varying length extending from one pole as a continuation of the long axis of the cell (Fig. 1). At the time of separation one cell possess a prostheca and the other a single polar agellum. Based on the phenotypic characteristics (Table 1), it can distinguish strain LZ-16-1 T from other type strains of the genus Maricaulis. Strain LZ-16-1 T grew at a temperature range of 10-40°C and a pH range of 5.0-9.0 ( Table 1). The optimal conditions turned out to be pH 7.0 and 25-30°C. The observed optimum temperature of LZ-16-1 T is lower compared to M. parjimensis MCS 25 T (30-40°C) and M. virginensis VC-5 T (20-40°C).

Comparative genomic characteristics
Due to the high 16S gene sequence similarity between strains LZ-16-1 T and M. parjimensis MCS 25 T , the whole-genome of the type strain of M. parjimensis was sequenced and submitted to GenBank with the accession no. JAEMQH000000000. As shown in Table 3, the draft genome size of strain M. parjimensis MCS 25 T was 3,348,930 bp with 17 contigs with an N 50 of 406 kb. It has 3,157 protein-coding genes with the DNA G+C content of 62.9 mol% calculated from the genome. The genome of LZ-16-1 T has a size of 3,348,699 bp which is very close to M. parjimensis (3,348,930 bp), whereas the GC content of strain LZ-16-1 T (63.6%) is slightly higher. Automated gene prediction and annotation identi ed 3,146 putative protein-encoding genes, of which 26.04% (834 genes) are annotated as hypothetical proteins. For strains LZ-16-1 T and M. parjimensis MCS 25 T , total 2,286 genes (72.6%) and 2,263 genes (73.0%) were functionally annotated within COG database, respectively (Fig. S4, pane a). However, only 1,486 (47.2%) and 1,205 genes (38.9%) were annotated by KEGG for metabolic pathways, indicating that a number of functional genes were still indistinct (Fig. S4, pane b). In addition, strain LZ-16-1 T showed lesser number of genes encoding for carbohydrate-active enzymes (CAZymes) compared to strain M. parjimensis MCS 25 T (Fig. S4, pane c). Similar pro le was also observed for the comparison of gene annotation by COG database which resulted in 27 functional categories (Fig. S3, pane b), although the two strains demonstrated only 231 bp difference between their genomic sizes.
In order to identify the multiple maximal matches and local collinear blocks (LCBs), a multiple wholegenome alignment of genome sequences of strains LZ-16-1 T and M. parjimensis MCS 25 T were performed using PATRIC software (www.patricbrc.org). The LCBs alignments of two strains resulted in 34 rearranged pieces larger than 1 kb, and it demonstrated obviously difference from each other (Fig. S5). It indicates a clear-cut genomic dissimilarity between the two strains. Moreover, the phylogenomic calculations of ANI and dDDH values between strains LZ-16-1 T and M. parjimensis MCS 25 T were only 85.0 and 20.9%, respectively (Table 1). Both values were clearly far below the threshold values for species delineation (Chun et al. 2018). Therefore, it strongly indicated that strain LZ-16-1 T represents a novel species of the genus Maricaulis.
Based on the genome annotation, the discrete biosynthetic components for holdfast polysaccharide synthesis, agellar motility and type II pilus assembly were identi ed in the genome of strain LZ-16-1 T (Table S1). EPS has been revealed to serve as one vital chemical intermedia within microscopic phycosphere niches, and mediates the host-microbe interactions involving in those cross-kingdom exchanges of nutrients, infochemicals and gene transfer agents (Amin et al. 2012;Seymour et al. 2017;Zhang et al. 2020). Remarkably, the bio occulanting activity of EPS produced by strain LZ-16-1 T was discovered by our bioactivity assay (Fig. S1) (Mu et al. 2019). Correspondingly, series of genes (wza, exo and muc) responsible for bacterial EPS biosynthesis were found in the genome of strain LZ-16-1 T (Table   S1). Thus, we proposed that strain Z10-6 T could serve as a novel bacterial candidate with natural potential for the production of promising and versatile bio occulants (Duan et al. 2020;Yang et al. 2020;Zhang et al. 2020).

Taxonomic conclusion
Based on the polyphasic evidences by phenotypic characterization, phylogenetic and genome comparison, and chemotaxonomic analysis, strain LZ-16-1 T clearly represents a novel species of the genus Maricaulis, for which the name Maricaulis alexandrii sp. nov. is proposed.
Maricaulis alexandrii (a.le.xan'dri.i. N.L. gen. n. alexandrii of the dinoglagellate Alexandrium catenella, the source of the isolation of the type strain).
The type strain, LZ-16-1 T (=CCTCC AB 2018386 T =KCTC 72198 T ), was isolated from the cultivable phycosphere microbiota of highly-toxic harmful algal blooms dino agellate Alexandrium catenella LZT09, which was collected in Zhoushan Archipelago ares in the East China Sea during an algal bloom occurred in July of 2018, and then routinely cultured in ABI Laboratory. The GenBank/EMBL/DDBJ accession numbers for 16S rRNA gene sequence and draft genome sequence of strain LZ-16-1 T are MK100326 and SWKP00000000, respectively.

Electronic Supplementary Materials
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-Con icts of interest/Competing interests
The authors declare that there are no con icts of interest.
-Ethics approval (include appropriate approvals or waivers) Not applicable -Consent to participate (include appropriate statements) Not applicable -Consent for publication (include appropriate statements) All authors have read and approved the manuscript.
-Availability of data and material (data transparency) Strain LZ-16-1 T has been deposited in two culture centers (CCTCC in China, and KCTC in South Korea) with the deposition no. CCTCC AB 2019006 T and KCTC 72194 T . The GenBank/EMBL/DDBJ accession numbers for 16S rRNA gene sequence of strain LZ-16-1 T is MK100326, and for draft genome sequences of strains LZ-16-1 T and Maricaulis parjimensis MCS 25 T are SWKP00000000 and JADOTT000000000, respectively.
-Code availability (software application or custom code)

Figure 2
The constructed neighbour-joining (NJ) phylogenetic tree showing the phylogenetic position of strain LZ-16-1T and representatives of other related taxa based on 16S rRNA gene sequences. Filled circles indicate nodes that were also recovered in the maximum-parsimony (MP) tree and the maximum-likelihood (ML) tree based on the same gene sequences. Bootstrap values (expressed as percentages of 1000 replications) > 50% are shown at branching points for NJ/ML/MP trees. Rhodovulum phaeolacus JA580T was used as an outgroup. Bar, 0.01 nt substitution rate (Knuc) units.

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