Photobacterium arenosum WH24, Isolated from the Gill of Pacific Oyster Crassostrea gigas from the North Sea of Germany: Co-cultivation and Prediction of Virulence

Cream colored bacteria from marine agar, strain WH24, WH77, and WH80 were isolated from the gill of the Crassostrea gigas a Pacific oyster with a filter-feeding habit that compels accompanying bacteria to demonstrate a high metabolic capacity, has proven able to colonize locations with changing circumstances. Based on the 16S rRNA gene sequence, all strains had high similarity to Photobacterium arenosum CAU 1568T (99.72%). This study involved phenotypic traits, phylogenetic analysis, antimicrobial activity evaluation, genome mining, Co-cultivation experiments, and chemical studies of crude extracts using HPLC and LC-HRESIMS. Photobacterium arenosum WH24 and Zooshikella harenae WH53Twere co-cultivated for 3 days in a rotary shaker at 160 rpm at 30 °C, and LC-MS monitored the chemical profiles of the co-cultures on the third day. The UV chromatograms of the extracts of the co-cultivation experiments show that Zooshikella harenae WH53T could be inhibited by strain WH24. The high virulence of Photobacterium arenosum WH24 was confirmed by genome analysis. Gene groups with high virulence potential were detected: tssA (ImpA), tssB (ImpB/vipA), tssC (ImpC/vipB), tssE, tssF (ImpG/vasA), tssG (ImpH/vasB), tssM (IcmF/vasK), tssJ (vasD), tssK (ImpJ/vasE), tssL (ImpK/vasF), clpV (tssH), vasH, hcp, lapP, plpD, and tpsB family. Supplementary Information The online version contains supplementary material available at 10.1007/s00284-022-02909-2.


Introduction
Strains of the genus Photobacterium are facultative aerobes and belong to the class of gammaproteobacteria. These Gram-negative bacteria can be present in various places, including fish guts, surfaces, light organs, free-living bacteria in the aquatic water column, and rotting animal tissue. Based on their genetic makeup, Aliivibrio and Vibrio are closely linked genera to Photobacterium [1]. The genus was first described by Beijerinck [2]. At the time of writing this article, the genus Photobacterium comprises 37 taxa with a validly published and correct name (https:// www. bacte rio. net/). Because of their luminous activity and use as a biosensor agent [3] and their ability to produce polyunsaturated fatty acids [4], antibacterial compounds [5], lipases [6], asparaginases [7], and esterases [8], Photobacterium species have been identified as an important group of bacteria. Humans can become sick by swallowing infected fish or bathing in brackish water when Photobacterium species enter person's urinary tract. Organ dysfunction, necrotizing fasciitis, and even death may occur in humans. Humans can tolerate the pathogen for up to 72 h. Antibiotics and radiation have both been used and attempted to cure the infection. Amputating the affected region of the body until the pathogen spreads is the safest option [9]. The majority of species are found in the light organs of various marine animals, including fish and squid. As pathogens or decomposers of deceased fish and commensals in the guts of many marine creatures, this form of connection might be characterized as symbiotic growth in the light organs of fish and squid [10]. However, it is known that luminous bacteria are widely distributed in marine habitats and can be found both free-living and host-associated. Photobacterium, cal, and signal diversity of QS has advanced dramatically in recent years [14]. Bacteria employ a variety of techniques to interact with their surroundings and hosts. The majority of these interactions are dependent on protein synthesis and secretion. Protein secretion in bacteria is governed by various processes that are rather complicated and dependent on the organism's structure. As a result of changes in the structure of the bacterial cell walls and the bacterial cell membrane, this mechanism will be unique in positive and negative gram bacteria. Despite extensive study and significant progress in understanding secretion systems, the method and the structural and molecular mechanisms of these systems remain unknown. Specific secretion systems are a prerequisite for quorum sensing, so it is even more important to understand their individual components and how they work [15]. Type I to Type VI secretion systems (T1SS-T6SS) are known in Gram-negative bacteria. Different components, substances, and processes are found in each system. Materials must flow through both the inner and outer membranes of these bacteria, or specific compounds must enter the host cell, necessitating the use of a variety of molecules and processes [16]. Single-stage paths are Systems I, III, IV, and VI. These systems deliver the molecules they transport directly into the extracellular space without passing the periplasm. In two-stage secretion systems like II and V, proteins enter periplasm space with the aid of general secretion systems like Tat and Sec to find the appropriate folding and then find their way out via one of the two-stage secretion systems in the second phase [15].
One of the virulence factors in many pathogenic bacteria is The patatin-like protein D (PlpD) prototype of the subclass T5dSS, which secretes a lipolytic passenger that forms extracellular homodimers. This enzyme is released to a 16-stranded -barrel transporter, comparable to TpsB transporters seen in type Vb secretion systems [17].
Marine-derived bacteria have promise as a source of new bioactive chemicals, crucial for therapeutic development. However, like with terrestrial microbes, there is a high redundancy rate, resulting in the regular re-discovery of known chemicals. Under typical laboratory circumstances involving the development of axenic microbial strains, only a portion of the biosynthetic genes encoded by bacteria seems to be translated. Furthermore, most biosynthetic genes are not expressed in vitro, limiting the chemical variety of microbial chemicals created by fermentation. Co-cultivation (also known as mixed fermentation) of two or more distinct microbes, on the other hand, attempts to simulate the biological condition in which germs co-exist in complex microbial communities. During co-cultivation, the competition or antagonism results in greatly increased synthesis of constitutively present chemicals and/or a buildup of cryptic compounds not identified in the generating strain's axenic cultures [18]. Although this article does not address the pathogenicity of humans, we mentioned some important genomes with virulence effects. We isolated the Photobacterium species (WH24, WH77, and WH80) related photobacterium arenosum and analysed some elements that have an important role from ecological aspects, their secondary metabolite productions, co-cultivation, and important properties extracted from genome mining studies.

16S rRNA Gene Analysis
The Invisorb Spin Plant Mini Kit was used to extract genomic DNA following the instruction of the kit manufacturers (Stratec Molecular, Germany). PCR amplification of the 16S rRNA gene was performed according to Primahana et al. [25] with the primer F27 (5′AGA GTT TGATCMTGG CTC AG3′) and 1492R (5′TAC GGY TAC CTT GTT ACG ACTT-3′) [26]. The 16S rRNA gene was sequenced employing an Applied Biosystems 3730XL automated sequencer (ABI). BioEdit software was used to modify and assemble the sequence (version 7.0.5.3) [27]. The 16S rRNA gene sequence of strains WH24, WH77, and WH80 were almost completely sequenced (1,415 bp) and submitted in Gen-Bank under the accession number MW888979, OM533648, and OM533649, respectively. The closest strains of strains WH24, WH77, and WH80 were identified based on 16S rRNA gene sequence similarity using the EZBioCloud system (https:// www. ezbio cloud. net/) [28]. Based on Blast analysis (https:// www. ncbi. nlm. nih. gov/) using 16S rRNA gene sequence, it was found that the 16S rRNA gene sequences of strain WH24 (1415 bp), WH77 (1343 bp), and WH80 (1262 bp) were 100% identical. Therefore, for further study, one of the strains (WH24) was used for evaluating its phenotypic and genotypic properties. Phylogenetic analysis of the 16S rRNA gene of strain WH24 with the closely related type strains was inferred by GGDC online server (http:// ggdc. dsmz. de/) [29]. The sequence was analysed using a single-gene adaptation of the DSMZ phylogenomics program [30]. Multiple sequence alignment was performed using MUSCLE [31]. Randomised Axelerated Maximum Likelihood (RAxML) [32] and TNT (Tree analysis using New Technology) [33] programs were used to estimate Maximum likelihood (ML) and Maximum parsimony (MP) trees, respectively. For ML analysis, we used rapid bootstrapping with the autoMRE (extended majority rule) bootstrapping criteria [34]. In the case of MP, 1000 bootstrapping replicates were employed, and tree bisection and reconnection branch switching and ten random sequence addition repetitions. The X 2 tests used in PAUP* (Phylogenetic Analysis using Parsimony*) were used to analyse the sequences [35].

Chemotaxonomy
The biomass used in the chemical analyses was grown and collected for 7 days at 30 °C in a 250-mL flask containing 100 mL MB medium on a rotary shaker (160 revolutions per minute). The chemotaxonomic analysis was conducted on freeze-dried biomass. Minnikin's technique [36] for obtaining isoprenoid quinones was adopted. The compounds were analysed using high-performance liquid chromatography fitted with diode-array detection and mass spectrometry (HPLC-DAD-MS) described by Risidian et al. [37], with some adjustments to the column, mobile phase, and flow rate. For isocratic conditions, solvent A (35% isopropanol + 1 percent water + 0.1% formic acid) and solvent B (65% acetonitrile + 1% water + 0.1% formic acid) were utilized at a flow rate of 0.3 mL/min. The isoprenoid quinones were separated using a Waters ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 m). The extraction and methylation of fatty acids were carried out in accordance with Sasser's protocol [38]. Fatty acid methyl esters (FAME) were analysed using an Agilent 6890N gas chromatography fitted with a flame ionization detector (FID). The methyl esters of fatty acids were isolated using a Macherey Nagel Optima 5 column (5% phenyl, 95% dimethylpolysiloxane; 50 m length; 0.32 mm inner diameter; 0.25 m film thickness). Their retention periods were compared to standards (in-house reference standard) to identify specific fatty acid methyl esters.
The draft genome of strain WH24 was submitted to DDBJ/EMBL/GenBank with the accession number JAGSOZ000000000.

Secondary Metabolite Production and Antimicrobial Activity
Growth of the strain WH24 was carried out for 5 days at 30 °C on a shaker in 250-ml Erlenmeyer flasks that contained 100 mL of MB medium with 2% (v/v) XAD-2 polymeric resin (160 revolutions per minute). Separation of XAD-2 was performed using a paper filter, and acetone was employed to extract secondary metabolites from the XAD-2. The extract was dried at 40 °C in a rotary evaporator. The dried extract was diluted in 1 mL methanol and evaluated for antimicrobial activity against a variety of bacteria:

Co-culture Experiment
In this study, we co-cultivated the strains that were isolated in the same location in Oyster for instance, Photobacterium sp. strain WH24 and Zooshikella harenae WH53 T were cultivated [53]

Base Peak Chromatogram Analysis of an Extract of Strain WH24
The analysis of the extract of strain WH24 was performed using an Agilent 1260 series HPLC-DAD system coupled with a MaXis ESI-TOF (Time of Flight) mass spectrometer (Bruker Daltonics, Bremen, Germany). The column C18 Acquity UPLC BEH (Ultra Performance Liquid Chromatography Ethylene Bridged Hybrid, Waters) was used as the stationary phase. The separation was carried out by gradient system employing two mobile phases (solvent A: H 2 O + 0.1% formic acid; solvent B: ACN + 0.1% formic acid) with the condition: 5% B (0.5 min), 5-100% B (0.5-20 min), and 100% B (20-25 min) and the flow rate was 0.6 mL/min (40 °C). Molecular formulas of the compounds were analysed using the Smart Formula algorithm (Bruker Daltonics) [54]. The selection of the major peaks of the base peak chromatogram (BPC) (cut-off intensity of 20%) was determined from the retention time of 1.5-18 min. The Dictionary of Natural Products database (DNP on USB, version 30.1, CRC Press, Taylor & Francis, Boca Raton, FL, USA) was used to predict the compounds based on the accurate mass with ± 0.01 Da.

Results and Discussion
Microscopic observations show that strain WH24 is Gramstain-negative and motile. The bacterium has single polar flagella and is rod-shaped with a cell size diameter of 0.5-0.8 µm in width and 1.5-2.6 µm in length (Supplementary Fig. S1). It seems to be an asporogenous bacterium. The optimal temperature for growth was determined to be 30 °C, and the optimal pH value was determined to be 7. On an agar medium without NaCl, low growth was identified. Tolerance to sodium chloride was up to 10%, with optimal growth occurring on conditions containing 2.5 and 5% sodium chloride. Biochemical properties based on Api ZYM, Api Coryne, and Api 20E assays that distinguish strain WH24 from its closest relatives are listed in Table S1, Table S2 and in the description of Photobacterium arenosum. All negative traits from commercial kits Api ZYM, Api Coryne, and Api 20E for Photobacterium arenosum WH24 are listed in Table S3. Strain WH24 could utilize mannitol, fructose, and cellulose as the sole carbon source. Strain WH24 was sensitive to polymyxin, gentamycin, chloramphenicol, thiostrepton, and erythromycin. However, the isolate was resistant to oxytetracycline, ampicillin, spectinomycin, kanamycin, cephalosporin, fusidic acid, bacitracin, trimethoprim, and tetracycline. According to Blast analysis, the 16S rRNA gene sequence of strain WH24 shows high similarity to Photobacterium arenosum CAU 1568 T (99.72%), Photobacterium salinisoli LAM9072 T (97.95%) and Photobacterium halotolerans MACL01 T (97.55%). In the phylogenetic tree, strain WH24 was located in the same clade with P. arenosum CAU 1568 T with a very high supported branch (Fig. 1). Based on whole-genome analysis, strain WH24 and Photobacterium arenosum CAU 1568 T had ANI values of 98.96% and dDDH scores of 90.8%, which are more than the species cut-off value of 95% and 70%, respectively [55]. Therefore, strain WH24 belongs to species Photobacterium arenosum. The major fatty acids identified in strain WH24 were C16:0 (21.79%), C16:1ω7c (17.11%), and C18:1ω7c (15.32%). The main polar lipids of strain WH24 were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), unknown aminophospholipid (APL), unknown phospholipids (PL), and unknown polar lipid (L) (Supplementary Fig. S2). The major quinone of strain WH24 was ubiquinone-8 (Q-8). Fig. 1 Phylogenetic tree based on 16S rRNA gene sequence of strain WH24 and type strains of the closely related species of the genus Photobacterium. The GTR + GAMMA model was used to infer the ML tree, which was then rooted using midpoint-rooting. The branches were scaled in terms of the expected number of substitutions per site. The numbers above the branches are support values when larger than 60% from ML (maximum likelihood, left) and MP (maximum parsimony, right) bootstrapping. The ML bootstrapping did not converge; hence 1000 replicates were conducted; the average support was 72.46%. MP analysis yielded the best score of 502 (consistency index 0.66, retention index 0.60) and 2 best trees. The MP bootstrapping average support was 86.38% The draft assembled genome sequence of strain WH24 consisted of 4,645,931 bp with a G + C content of 50.18% (GenBank accession No. JAGSOZ000000000). The genome included 4270 genes comprising 4181 protein-coding genes, 81 tRNA genes, 4 rRNA genes, and 4 non-coding RNA. According to the phylogenomic tree (Fig. S3), strain WH24 formed a clade with Photobacterium arenosum CAU 1568 T . The results of genome mining by the Whole-genome showed just one difference for T6SS effector tse gene for strain WH24 isolated from Pacific Oyster from Germany and Photobacterium arenosum CAU 1568 T . Other comparisons are determined and listed in Table 1. Based on RAST analysis, it was discovered that 28% of the genes were allocated to subsystems (Fig. 2). The largest number of predicted gene clusters concerned the metabolism of amino acids and derivatives (326), followed by protein metabolism (208) and carbohydrate metabolism (186). Genes responsible for motility and chemotaxis (110), metabolism of aromatic compounds (11), stress response (88), and dormancy and sporulation (4) were also detected. Eleven gene clusters involved    Table 2, it can be seen that the microbial activity of the extract derived from the co-cultivation mostly followed the bioactivity pattern of the extract from WH24. The UV chromatograms of the co-cultivation extracts also confirmed that peaks of Zooshikella harenae WH53 T [53] extracts could not be seen, suggesting that the growth of Z. harenae WH53 T [53] might be inhibited by strain WH24 (Fig. 3 and Table. S4). Interestingly, a high peak was detected in the extract from co-cultivation of the strains. The peak was identified at 3.   Whoever, it can be concluded that this type of bacterium can be a potential cause of disease in marine animals, as possible as the presence of Quorum-sensing regulator of virulence hapR, luxR family, and other genes might control gene expression in response to changes in cell population density. On the other hand, analysis of the Co-culture experiment for this study showed that these bacteria would produce new secondary metabolites and inhibit the target bacteria if they grow together. The UV chromatograms of the co-cultivation extracts also confirmed that Zooshikella harenae WH53 T could be inhibited by strain WH24. Therefore, further study on these bacteria as well as their interaction with marine organisms in the aquatic environment is recommended.