Hyalangiumruber sp. nov, characterization of a novel myxobacterium strain s54d21 and their secondary metabolites

Myxobacteria are special bacteria with wide adaptability, which are rich sources of structurally diverse natural products with intriguing biological properties. Here, a gram-negative myxobacterium strain s54d21T was isolated from the sediment of a wetland park in China using the Escherichia coli baiting method. Based on 16S rRNA gene sequence and genomic data, the strain was demonstrated to be a novel species of a rare genus Hyalangium, designated Hyalangium ruber sp. nov (type strain s54d21T = GDMCC 1.1945T = JCM 39263T). The subsequent chemical investigation of the strain s54d21T led to the isolation of three rare 3,5,6-trisubstituted 2(1H)-pyrazinones, namely, hyalanones A–C (1–3), together with a known macrolactin A (4). Those new structures and their absolute configurations were unambiguously assigned by extensive analyses of spectroscopic data and density functional theory (DFT) calculations. In biological assays, compound 4 exhibited moderate cytotoxic activities against human cell lines RKO, A549, and NCM460 with IC50 values ranging from 27.21 to 32.14 μM.


Introduction
Myxobacteria are a class of gram-negative eubacteria widely found in terrestrial and aquatic ecosystem.They are known for their complex lifecycle and social behavior that are probably mediated through secondary metabolites (Kaiser, 2003;Shrivastava and Sharma, 2021;Saggu et al., 2023).In past four decades, numbers of natural products from myxobacteria have been characterized with novel and complex structures, including polyketides, non-ribosomal peptides, and hybrids (Wenzel and Müller, 2009;Herrmann et al., 2017;Bader et al., 2020;Saggu et al., 2023).These secondary metabolites exhibit diverse biological properties, such as cytotoxic, antifungal, antibacterial, antiviral, antimalarial, and immunosuppressive effects (Wenzel and Müller, 2009;Schäberle et al., 2014;Dehhaghi et al., 2018;Bhat et al., 2021).Hence, myxobacteria are considered as important and enormous sources for structurally diverse and bioactive natural products that can be explored for drug discovery.Zang et al. 10.3389/fmicb.2024.1369499Frontiers in Microbiology 02 frontiersin.org Although amount of myxobacteria have been identified in past two decades, there are numerous undiscovered or uncultivated myxobacteria.The genus Hyalangium belonging to Myxococcaceae family was initially established by Reichenbach (2005), while the only member H. minutum was isolated from a mountain soil with decayed plants before 2023.Continuous chemical research studies of H. minutum found a series of bioactive compounds, including hyaladione with significant cytotoxic and antimicrobial activities and unusual siderophores, namely, hyalachelins A-C (Okanya et al., 2012;Nadmid et al., 2014;Okanya et al., 2014;Surup et al., 2018).Until recently, two novel species of H. versicolor and H. gracilis were characterized and reclassified by Zhang et al. (2023).
In this study, the strain s54d21 T was isolated from a sample of wetland sediment as a novel member of Hyalangium classified by using the polyphasic approach.To search for novel and bioactive natural products from this strain, the chemical investigation of the culture broth of Hyalangium ruber sp.nov was performed, leading to the identification of three 2(1H)-pyrazinone derivatives, namely, hyalanones A-C (1-3), along with a known compound macrolactin A (4).The planar structures and absolute stereochemistry of those new isolates were unambiguously established by extensive spectroscopic methods and theoretical electronic circular dichroism (ECD) calculations with time-dependent DFT (TDDFT) methods.Hyalanones A-C (1-3) were assigned as rare 3,5,6-trisubstituted 2(1H)-pyrazinone analogs that were probably biosynthesized from valine and alanine or threonine, respectively.The known compound 4 was determined by comparing of NMR data and HRESIMS spectrum with literatures (Gustafson et al., 1989;He et al., 2013).Herein, we report the identification of the myxobacterium strain, isolation and structure elucidation of new compounds, and their biological assay.

Isolation and cultivation of the myxobacteria strain
Strain s54d21 T was isolated from a sediment sample collected from Xinghu National Wetland Park (N 23°4′54″, E 112°28′17″) in Guangdong Province of China.The strain was isolated following the baiting approach with E. coli as prey on water agar (0.1% CaCl 2 •2H 2 O, 20 mM HEPES, 1.5% agar), where small portions of soil samples were placed adjacent to the E. coli spot.Until swarming colonies or fruiting bodies observed after incubation at 30°C, purification of the isolate was subsequently completed by repeatedly transferring onto fresh VY/2 agar (0.5% dried baker's yeast, 0.1% CaCl 2 •2H 2 O, 1.5% agar).Finally, the pure strain characterized orange-pigment fruiting bodies on agar and was deposited in Guangdong Microbial Culture Collection Center (GDMCC, No. 1.1945) and Japan Collection of Microorganisms (JCM, no.39263).

16S RNA analysis identification and phylogenetic analysis
Genomic DNA of strain s54d21 T was extracted by using Genomic DNA Isolation Kit (Magen, China).The 16S rRNA gene sequence was amplified using the universal primers 27F/1492R (Weisburg et al., 1991).The PCR products were sequenced by Shanghai Majorbio Biopharm Technology Co., Ltd.Sequence alignment was performed at online EZBioCloud server (Yoon et al., 2017a) and NCBI.Phylogenetic tree based on the 16S rRNA gene sequences was reconstructed using software MEGA X (Kumar et al., 2018) with the maximum likelihood (ML) method (Felsenstein, 1981) under the Kimura's two-parameter model (Kimura, 1980).Bootstrap analysis was conducted based on 1,000 replicates (Hoang et al., 2018).

Genome sequencing and bioinformatics analysis
Genomic sequencing was carried out using an Illumina Hiseq platform at Shanghai Majorbio Biopharm Technology Co., Ltd.The obtained reads were assembled into contigs using SPAdes v3.11.1 (Bankevich et al., 2012).Genome contamination and completeness were assessed using CheckM tool (Parks et al., 2015).Genome was annotated using the Prokka (Seemann, 2014) and RAST server (Aziz et al., 2008).The biosynthetic gene clusters of secondary metabolites Zang et al. 10.3389/fmicb.2024.1369499Frontiers in Microbiology 03 frontiersin.orgwere predicted using antiSMASH 6.0 with the default settings (Blin et al., 2021).The carbohydrate activities were identified using the dbCAN2 meta server (Zhang et al., 2018).Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values among strain s54d21 T and the closely related type strains were calculated using an online ANI calculator and the Genome-to-Genome Distance Calculator 3.0 (Meier-Kolthoff et al., 2013;Yoon et al., 2017b).Phylogenomic tree was constructed using the up-to-data bacterial core gene (UBCG) pipeline based on 92 core housekeeping genes (Na et al., 2018).

Chemotaxonomic characterization
Respiratory quinones of the strain s54d21 T were extracted and analyzed as previously described using HPLC (Agilent 1260) (Collins et al., 1977).For the fatty acid detection, strain s54d21 T and its closely related type strains were incubated on VY/2 agar plates at 30°C for a week.Fatty acids were extracted and identified according to the described protocol using a gas chromatography mass spectrometry (GCMS, Agilent 7890B-5977B) (Gemperlein et al., 2014).

General experimental procedures
The UV, IR, and ECD spectra were obtained on a Jobin Yvon LabRAM HR800 instrument, a Bruker Vertex 70 instrument, and a JASCO-810 ECD spectrometer, respectively.Optical rotation values of new compounds were tested with a Rudolph Autopol IV automatic polarimeter.NMR spectra were recorded on a Bruker Ascend 600 MHz spectrometer, with 1 H and 13 C NMR chemical shifts referenced to the solvent or solvent impurity peaks for CD 3 OD (δ H 3.31 and δ C 49.0).HRESIMS data were acquired using electrospray ionization (ESI) with a Thermo Fisher Orbitraq Exploris 120 mass spectrometer.Semi-preparative HPLC utilized a SHIMADZU Prominence LC-20AT quaternary system with a UV-VIS detector using a YMC-pack ODS-A column (5 μm, 10 × 250 mm).Column chromatography (CC) was performed using ODS (50 μm, YMC, Japan).Thin-layer chromatography (TLC) was performed with silica gel 60 GF254 (Yantai Chemical Industry Research Institute, China)., extraction, and isolation   2.6.2

.1 Extraction and isolation
The strain was maintained on VY/2 agar at 30°C for 5 days, which was used as the seed culture.The strain was cultured in 10 L MD1G liquid medium (0.3% tryptone, 5.0% starch, 0.02% MgSO 4 •2H 2 O, 0.07% CaCl 2 •2H 2 O) on a rotary shaker (180 rpm) at 30°C for 10 days.Amberliter XAD-16 (2%, v/v) was added to the liquid medium on the fourth day (Okanya et al., 2012(Okanya et al., , 2014)).After incubation, XAD and cell mass were harvested by centrifugation and filtration, which was washed using 10 L acetone and 10 L methanol sequentially at room temperature to yield 600 mg of crude extract.

Theoretical computation method for structural identification
In general, conformational analyses were carried out via random searching in the Sybyl-X 2.0 (2013) using the MMFF94S force field with an energy cutoff of 5.0 kcal/mol.The results showed 15 lowest energy conformers.Subsequently, geometry optimizations and frequency analyses were implemented at the B3LYP-D3 (BJ)/6-31G* level in CPCM acetonitrile using ORCA5.0.1 (Neese, 2012(Neese, , 2017)).All conformers used for property calculations in this study were characterized to be stable point on potential energy surface (PES) with no imaginary frequencies.The excitation energies, oscillator strengths, and rotational strengths (velocity) of the first 60 excited states were calculated using the TD-DFT methodology at the PBE0/def2-TZVP level in CPCM acetonitrile using ORCA5.0.1.The ECD spectra were simulated by the overlapping Gaussian function (half the bandwidth at 1/e peak height, sigma = 0.30 for all) (Stephens and Harada, 2010).Gibbs free energies for conformers were determined by using thermal correction at B3LYP-D3 (BJ)/6-31G* level, and electronic energies were evaluated at the wB97M-V/def2-TZVP level in CPCM methanol using ORCA5.0.1.To get the final spectra, the simulated spectra of the conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy (∆G).

Cytotoxicity assay
The cytotoxicity was determined by the MTS method as reported in our previous studies (Zang et al., 2020(Zang et al., , 2022)).Colorimetric assays were performed to evaluate the activity of each compound.A549 (human lung cancer cells), HepG-2 (human hepatocellular carcinoma cells), MCF-7 (human breast adenocarcinoma cells), RKO (human colon cells), and NCM460 (normal human colon mucosal epithelial cells) were purchased from the National Collection of Authenticated Cell Cultures of China.All cells were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (Hyclone) at 37°C in a humidified atmosphere with 5% CO 2 .Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction in 3-(4,5-dimethylthiazol-2-yl)-5(3carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium (MTS) (Sigma, St. Louis, MO).In brief, 100 μL of suspended adherent cells were seeded into each well of a 96-well cell culture plate and allowed to adhere for 12-24 h before the addition of a drug.In addition, suspended cells were seeded just before the addition of a drug, with an initial density of 1 × 10 5 cells/ml in 100 μL of medium.Each cell line was exposed to each test compound at various concentrations in triplicate for 48 h.After the incubation, MTS solution (20 μL) was added to each well, and the incubation was continued for 2-4 h at 37°C.The optical density of the lysate was measured at 492 nm in a 96-well microtiter plate reader (MULTISKAN FC).The IC 50 value of each compound was calculated by Reed and Muench's method.

Antimicrobial assay
The antimicrobial effect was determined following the method as reported in the previous study (Zang et al., 2016).Reference strains of Staphylococcus aureus (ATCC43300), Escherichia coli (ATCC11229), Pseudomonas aeruginosa (ATCC9027), and Enterococcus faecalis (ATCC29212) were cultivated for 24 h in Luria-Bertani medium (LB) at 37°C.Precultures of the tested microorganisms were made by inoculating 50 mL of LB medium and incubated for 24 h at 37°C.A culture suspension was made by 1/1000 dilution (OD600 0.01) from preculture and seeded in 96-well microtitration plates.In total, 1 μL of two-fold serial dilutions of each compound (40 mM) was prepared in 200 μL of medium.After 24 h, the optical density of the bacterial suspension of each well was measured at 595 nm using a multiplate reader (MULTISKAN FC).The MIC, which is the minimal concentration of a compound resulting in a 90% decrease in the number of microbial cultures compared with a control (DMSO only), was determined by curve fitting for bacteria.Vancomycin (Sigmaaldrich, Merck) was used as positive control.

Anti-inflammatory assay
RAW 264.7 cells were obtained from National Collection of Authenticated Cell Cultures (Shanghai, China) and maintained in DMEM containing 10% fetal bovine serum (FBS) (Gibco BRL Co, Grand Island, NY, United States) at 37°C in humidified incubator containing 5% CO 2 .All tested compounds were dissolved in DMSO (the final concentration of DMSO was less than 0.25% in assay).Cell viability was evaluated by the CCK-8 method after 24 h of incubation with test compounds of 40 μM.RAW 264.7 cells were seeded into 96-well plates (2.5 × 10 5 cells/well) for 24 h and then pretreated with test compounds.After being incubated for 3 h, the cells were stimulated with 100 ng/mL LPS (final concentration) for another 24 h.Celastrol (Sigma-aldrich, Merck) was used as the positive control.NO content in the supernatant was measured using Griess reagent.The absorbance at 540 nm was measured on a microplate reader (MULTISKAN FC).

Phylogenetic analysis based on 16S rRNA gene sequence
The nearly complete 16S rRNA gene sequence of the strain s54d21 T was obtained with the length of 1,536 bp (GenBank accession number: OR885464).Pairwise comparison of 16S rRNA gene revealed that the strain s54d21 T shared the highest similarity to H. minutum DSM 14724 T (98.2%), followed by H. versicolor H56D21 T (97.8%) and H. gracilis DSM 14753 T (97.7%).Phylogenetic analysis based on 16S rRNA gene sequence showed that the strain s54d21 T located in the genus Hyalangium and formed an individual branch containing all type strains of Hyalangium genus (Figure 1).

Genome sequencing and phylogenomic analysis
The draft genome of the strain s54d21 T was 10.77 Mb in size with 43 contigs, the N50 of 586,569 bp, and the DNA G + C content of 68.5 mol% (Supplementary Table S1).The genome contained 8,621 protein-coding sequences, 70 tRNAs, and 3 rRNAs genes.The UBCGbased phylogenomic tree showed that the strain s54d21 T was closely related to all type species of Hyalangium (Figure 2), which was consistent with the tree topology based on the 16S rRNA genes.The calculated genome similarity values between the strain s54d21 T and other type species in the genus Hyalangium were in the ranges of 81.3-82.7% for ANI and 24.4-25.7%for dDDH (Supplementary Table S2), which were all below the threshold values for species delineation (95.0-96.0%for ANI and 70.0% for dDDH) (Goris et al., 2007;Richter and Rosselló-Móra, 2009).These results suggested that the strain s54d21 T should be considered as a novel species in the genus Hyalangium, for which the name Hyalangium ruber sp.nov was proposed.
The RAST annotation result showed that almost 81.0% of coding sequences in the strain s54d21 T were not assigned to subsystems.The general feature of subsystem category distribution in the strain s54d21 T genome was similar to those in its closest phylogenetic neighbor.No genes involved in motility and chemotaxis were found to be encoded by the strain s54d21 T genome and in the genomes of closely related type strains.However, genes associated with iron acquisition and metabolism were found in the strain s54d21 T more than these in the three closely related strain genomes (Supplementary Figure S1).Based on CAZy database annotation, the genome of the strain s54d21 T contained 23 carbohydrate-binding modules, 33 carbohydrate esterases, 78 glycoside hydrolases, 61 glycosyl transferases, 6 polysaccharide lyases, and 15 auxiliary activities (Supplementary Figure S1).There were most types of carbohydrate enzymes involved in glycoside hydrolases, which can

Description of Hyalangium ruber sp. nov
Hyalangium ruber (ru′ber.L. masc.Adj.ruber red, referring to the color of the colonies).
The type strain s54d21 T (= GDMCC 1.1945 T = JCM 39263 T ) was isolated from the sediment sampled from Xinghu National Wetland Park located in Guangdong Province, China.The GenBank accession numbers for the 16S rRNA gene and whole genome sequences of the type strain are OR885464 and JAXIVS000000000, respectively.

Structure elucidation of new compounds
The gene sequence analyses of the strain s54d21 T supposed the production of polyketides and peptides in terms of the presence of PKS and NRPS BGCs.Therefore, the chemical investigation of the culture broth of the strain was performed as described.Here, the  2) and HSQC spectrum presented signals of ten carbons, including four methyls (a singlet at δ H 2.22, two doublets at δ H 1.20, and a triplet at δ H 1.17), a methylene at δ H 2.57 (q, J = 7.5 Hz) and δ C 26.1, a methine at δ H 3.34 (m) and δ C 30.9, and four unprotonated carbons. 1 H-1 H COSY correlations demonstrated the existence of two spin systems of an ethyl group and an isopropyl moiety (Figure 5).Combination analyses of the chemical formula and HMBC correlations (Figure 5) revealed that four unprotonated carbons (C-2, C-3, C-5, and C-6) and two nitrogens were assigned to the 2(1H)-pyrazinone ring.The comparison of those spectroscopic data suggested that structure of 1 was such close to those knowns featuring a 3,5,6-substitued 2(1H)-pyrazinone core structure (Motohashi et al., 2011;Ma et al., 2020).The differences between 1 and known compounds were three substituent groups located at C-3, C-6, and C-5, including an abovementioned ethyl group, an isopropyl group, and a methyl (Me-10), respectively.Subsequent analyses of HMBC spectrum supported those assignments for structure 1, where key HMBC correlations from H-7/Me-8 to C-2, H-7/Me-8/Me-10 to C-3, Me-10/ H-11 to C-5, and Me-10/Me-12 to C-6 were observed (Figure 5).Hence, compound 1 was unambiguously determined as a 3,5,6-trisubstituted 2(1H)-pyrazinone and designated as hyalanone A.
Hyalanone B (2) was also isolated as an amorphous solid.The molecular formula was demonstrated to be C 10 H 16 N 2 O 2 via a positive HRESIMS ion peak at m/z 219.1105 [M + Na] + .The 1 H and 13 C NMR data of 2 was such close to those of 1, except for an oxygenated methine at δ H 4.89 (q, J = 6.5 Hz, H-11) and δ C 66.6 (C-11) (Table 2), suggesting a similar 2(1H)-pyrazinone skeleton.A hydroxyethyl group replaced the ethyl at C-6 of 1, which was further confirmed by H-1 H COSY cross-peaks and key HMBC correlations from Me-12 to C-6/C-11 and H-11 to C-5 (Figure 5).There is not enough compound 2 for Mosher's method to determine the absolute configuration of the chiral center C-11.Therefore, it was inferred from theoretical ECD calculation by using TDDFT methodology at PBE0/def2-TZVP level in CPCM acetonitrile using ORCA5.0.1 (Neese, 2012(Neese, , 2017)).As shown in Figure 6, the experimental ECD curve of 2 was well matched with the calculated one that concluded the absolute configuration of C-11 to be R.Chemical structures of isolated compounds (1-4).Key 1 H-1 H COSY and HMBC correlations of compounds (1-3).The molecular formula C 11 H 18 N 2 O 2 of 3 was deduced from a HRESIMS spectrum.Analyses of 1D NMR data (Table 2) and HMBC correlations (Figure 5) of 3 exhibited a highly similarity with those of 2, where the unique difference between them was an extra methoxyl at δ H 3.22 and δ C 55.9 (11-OMe) in 3.Moreover, key HMBC correlation from 11-OMe to C-11 indicated the location of a methoxyl at C-11 of 3 rather than a hydroxyl in 2 (Figure 5).By comparing of experimental ECD curves of 2 and 3 (Figure 6), the absolute configuration of C-11 was still approved to be R. Therefore, compound 3 was established and given the name hyalanone C.
2(1H)-Pyrazinones are a type of natural products originally biosynthesized by NRPS gene cluster with two amino acids (Wyatt et al., 2010;Zimmermann and Fischbach, 2010;Wyatt et al., 2012).Based on biosynthetic pathways of pyrazinone analogs, three new compounds are proposed to be derived from the valine and alanine or threonine building blocks.Analyses of the genome sequence of strain s54d21 T by antiSMASH showed three NRPS-like and three PKS-NRPS BGCs.Among them, the NRPS (Region 1.3) and PKS-NRPS (Region 5.2) gene clusters containing a Val module are potent BGCs related to the biosynthesis of those new pyrazinones (Supplementary Figure S2).

Biological assays
The previous report of pyrazinone derivatives described their remarkable cytotoxicity against human cancer cell lines and antimicrobial activity (Jansen et al., 2014).Therefore, cytotoxic activities against human cancer cell lines and antimicrobial and antiinflammatory effects of all isolates were tested in this study.The cytotoxicity evaluation of compounds 1-4 was performed using the MTS method, where taxol was used as the positive control.Among them, compound 4 displayed moderate cytotoxicity against A549, RKO, and NCM460 with IC 50 values at 32. 14 ± 2.25, 27.31 ± 8.96, and 31.33 ± 1.71 μM (positive control with IC 50 values <0.008 μM), respectively.In addition, their inhibition of NO production in LPS-induced RAW 264.7 cells was tested.All compounds showed weak inhibitory effect (< 10.0%) of NO production at the concentration of 40 μM.The evaluation of their antimicrobial activities against S. aureus, E. coli, P. aeruginosa, and E. faecalis did not show obvious inhibition at the concentration of 200 μM.
Pyrazinones are previously reported to regulate phenotypic changes in the pathogen (e.g., quorum sensing) or act directly on the host (e.g., virulence factors), such as tyrvalin and phevalin are used as regulators of virulence factor gene expression (Wyatt et al., 2010;Secor et al., 2012).Thus, although three new pyrazinones do not exhibit obvious effects in the evaluation of cytotoxic, antimicrobial, and anti-inflammatory activities, the discovery of new molecules provides potential for further research studies of relationships between the small molecule and microorganisms as well as the role of myxobacteria in nature.

Conclusion
In this study, a novel myxobacterium s54d21 T belonging to a rare Hyalangium genus, designated Hyalangium ruber sp.nov, was purified from a wetland sediment using the E. coli baiting method.Uncovered by analysis of the gene sequence, the further chemical investigation of bioactive secondary metabolites from culture broth of H. ruber led to the production of three unprecedented 3,5,6-trisubstituted 2(1H)-pyrazinone derivatives, namely, hyalanones A-C (1-3), together with a known macrolactin A (4).In biological assay, compound 4 exhibited modest cytotoxic activities against human cell lines A549, RKO, and NCM460 with IC 50 values between 27.31 and 32.14 μM.Although three new compounds have not exhibited significant activities in biological assays, their structures enrich the chemical diversity of 2(1H)-pyrazinones from myxobacteria.Comparison of experimental and calculated ECD of 2 and 3. Zang et al. 10.3389/fmicb.2024.1369499Frontiers in Microbiology frontiersin.org

FIGURE 1
FIGURE 1Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences showing the relationships between strain s54d21 T and the closely related taxa.Bootstrap values were expressed as percentages of 1,000 replicates.More than 50% bootstrap values are shown.Bar, 0.02 the number of substitutions per site.The outgroup of the tree is Anaeromyxobacter dehalogenans 2CP-1 T .

FIGURE 3
FIGURE 3 Photographs showing colonies and cell morphology of the strain s54d21 T .(A) The stereoscopic photographs of swarming colonies on VY/2 agar after 5 days of incubation at 30°C (bar, 2 mm).(B) The transmission electron microscopic photographs of vegetative cells (bar, 5 μm).(C) The phase contract microscopic photograph of rod-shaped vegetative cells, spherical sporangioles (red arrow), and spherical myxospores (bar, 10 μm).

TABLE 1
Differential phenotypic and physiological characteristics of the strain s54d21 T and the closely related type strains.