A Gram-stain positive, aerobic, rod-shaped actinobacterial strain designated as JXJ CY 27-2T was isolated from the culture of Microcystis aeruginosa FACHB-905 (Maf) collected from Lake Kunming, southwest China. The isolate was catalase positive, oxidase negative, and able to grow at 10.0-44.0 °C, pH 5.0-10.0 and 0–5.0% NaCl. Based on the 16S rRNA gene sequences, JXJ CY 27-2T showed high similarities of 98.54–98.55% with Microbacterium invictum DSM 19600T, Microbacterium saccharophilum DSM 28107T, and Microbacterium aoyamense DSM 19461T, and less than 98.47% similarities with other members of the genus. Its major cellular fatty acids were anteiso-C17:0 and anteiso-C15:0. The predominant menaquinones were MK-11 and MK-12. The diagnostic diamino acid in the cell wall peptidoglycan was lysine. Whole cell sugars contained mannose, ribose, galactose, rhamnose and arabinose. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, two unidentified glycolipids, and an unidentified lipid. The DNA G + C content was 69.8%. The digital DNA-DNA hybridization and average nucleotide identity values between strain JXJ CY 27-2T and its three closest similar strains were 18.4–20.3% and 74.9–75.7%, respectively. Based on the above data, strain JXJ CY 27-2T was identified as a new species of the genus Microbacterium, for which the name Microbacterium kunmingensis sp. nov. is proposed. The type strain is JXJ CY 27-2T (=CGMCC 1.17506T = KCTC 49382T). Strain JXJ CY 27-2T could promote the growth of Maf by providing it with available phosphorus, nitrogen and probably other nutrients such as vitamins and indole-3-acetate.
The Lake Kunming (99°39’E, 22°23’N) also known as Lake Dianchi, the sixth largest freshwater lakes in China, is a plateau lake located in Yunnan-Guizhou plateau. Serious cyanobacterial blooms, mainly caused by Microcystis aeruginosa (Ma) , occurred almost every year from 1990 to 2005 in this lake mainly because of the dumping of agricultural, domestic and industrial waste . Cells of Microcystis were often covered by mucilages, which provide a special ecological niche for attached bacteria [3,4,5]. Interactions between the cyanobacteria and these bacteria are extremely complex. The interactions are categorized into nutrient exchange, signal transduction, and gene transfer, of which nutrient exchange has been considered as the most common type of interactions  and frequently the basis of cyanobacterial–bacterial mutualism . Cyanobacteria provide bacteria with dissolved organic materials, and bacteria provide cyanobacteria with various nutrients such as available P, N, vitamins  and plant growth hormone indole-3-acetic acid [9, 10]. However, the mutualism between cyanobacteria and bacteria is often mediated by nutrition supply from bacteria .
During a study of attached bacteria of M. aeruginosa FACHB-905 (Maf) collected from Lake Kunming, southwest China, a novel actinobacterium designated strain JXJ CY 27-2T, belonging to the genus Microbacterium, was isolated. This paper described the polyphasic characterization of the novel strain as well as the in vitro interactions between Maf and strain JXJ CY 27-2T in co-culture.
Materials and methods
Isolation, maintenance of strain
About 0.2–0.3 ml of Maf culture, obtained from Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB-collection), Chinese Academy of Sciences (Wuhan, China; http://algae.ihb.ac.cn/), was spread evenly on the surface of International Streptomyces Project 2 (ISP 2) agar plates  and incubated at 28.0 °C for 2–7 days to obtain pure cultures. Pure culture of strain JXJ CY 27-2T was stored on ISP 2 agar slants at 4.0 °C and in glycerol (30%, v/v) at −80.0 °C, respectively.
Phenotypic, physiological and biochemical characteristics
The cellular morphology was observed by using light microscopy (BX53; Olympus) and transmission electron microscope (TEM; JEM-2100, JEOL) after 3-day incubation on ISP 2 medium at 28.0 °C. The TEM specimen was prepared according to the method described previously . Gram staining was performed using the standard Gram’s staining procedure. Catalase activity was determined using H2O2 (3%) . Growth at different pH range (2.0–12.0 with interval of 1.0), temperatures (5.0, 10.0, 15.0, 20.0, 26.0, 28.0, 30.0, 32.0, 34.0, 36.0, 38.0, 40.0, 42.0, 44.0, 50 °C), and NaCl contents (0–10.0%, w/v, with interval of 1%) were performed using ISP 2 medium as the basal growth medium . Hydrolysis of starch, and Tweens (20, 40, 80), and H2S production were performed according to methods [15, 16]. The API systems were used to determine other physiological and biochemical characteristics according to the manufacturer’s instructions.
Biomass of strain JXJ CY 27-2T for chemical analysis was obtained by cultivation on ISP 2 agar medium at 28.0 °C for 3 days. Polar lipids were extracted and analyzed by two-dimensional thin-layer chromatography . Fatty acids were obtained, methylated and analyzed according to the standard procedure of the MIDI System (Sherlock version 6.1; database TSBA6). The menaquinone was detected using HPLC . Purified cell-wall was prepared and hydrolyzed according to Schleifer and Kandler . Cell-wall amino acids and sugars in cell-wall hydrolysates were analyzed by HPLC .
Phylogenetic and genotypic analysis
The genomic DNA preparation and PCR amplification of the 16S rRNA gene were performed as described previously . The almost-complete 16S rRNA gene sequence obtained was compared with available sequences of related valid species from EzBioCloud Databases (https://www.ezbiocloud.net/) . Phylogenetic trees were reconstructed by MEGA version 5.0  using the neighbor-joining method , and confirmed by maximum-parsimony  and maximum-likelihood  tree-making methods. Bootstrap analysis with 1000 replications was preformed to evaluate the topologies of the phylogenetic trees .
Genome sequencing and analysis
The whole genomes sequencing of strains JXJ CY 27-2T, Microbacterium invictum DSM 19600T, Microbacterium saccharophilum DSM 28107T and Microbacterium aoyamense DSM 19461T were performed using Illumina HiSeq 4000 platform at Sangon Biotech (Shanghai, China). The qualities of sequenced reads (raw reads) were evaluated using FastQC 0.11.2 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and handled using Trimmomatic 0.36 . The second-generation sequencing data were assembled using SPAdes 3.5.0  and corrected using PrinSeS-G 1.0.0 . Genetic factors prediction was performed using Prokka 1.10 . Repeat Region was determined using RepeatMasker 4.1.0. CRISPR prediction was performed using CRT 1.2 . Genomic annotation was carried out using NCBI Blast+ version 2.2.28 with the default parameters. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strain JXJ CY 27-2T and the related type strains were calculated by using the Genome-to-Genome Distance Calculator (version 3.0) (http://ggdc.dsmz.de/ggdc.php)  and JSpeciesWS website (http://jspecies.ribohost.com/jspeciesws/#analyse), respectively. The DNA G + C % value was calculated from the genomic sequences. The phylogenomic tree of strain JXJ CY 27-2T and some type strains of the genus Microbacterium was constructed on line by using the Type (Strain) Genome Server (https://tygs.dsmz.de/).
Abilities of nitrogen-fixing and dissolving unavailable phosphate
The ability of nitrogen-fixing was determined by culturing in medium of nitrogen-free  at 28 °C for 4 days, and the cell density was determined by spread plate counting. The ability of dissolving insoluble phosphorus was determined according to the method described by Zhang et al.  on day 2 of culture. Both Ca3(PO4)2, and phytin were used as the insoluble phosphorus at the dosages of 1 g l−1. All tests were set up in triplicates.
Co-culture of Maf and strain JXJ CY 27-2T
Maf was purified by streaking repeatedly on HGZ agar plates and purified Maf was cultivated in HGZ liquid medium . Maf (about 2 × 106 CFU ml−1) and strain JXJ CY 27-2T (final cell densities, 1 × 105, 1 × 106 and 1 × 107 CFU ml−1) were co-cultured as described previously  at 25 °C. Pure culture of Maf (about 2 × 106 CFU ml−1) was served as the control. Co-cultures and control were set up in triplicates and sampled on day 5, 10 and 15 of cultures. Cells densities of strain JXJ CY 27-2T in co-culture samples were examined using spread plate counting. The contents of chlorophyll a (chl-a), extracellular microcystin LR (E-MC-LR) and intracellular microcystin LR (I-MC-LR) were detected according to methods described previously .
Co-culture of Maf and JXJ CY 27-2T in absence of available P and N
Maf (about 2 × 106 CFU ml−1) and strain JXJ CY 27-2T (1 × 106 CFU ml−1) were co-cultured using revised HGZ media. In the revised HGZ media, KH2PO4 was replaced by Ca3(PO4)2, or NaNO3 was removed (nitrogen-free). Co-cultures and control were set up in triplicates. The cell densities of strain JXJ CY 27-2T, and the contents of chl-a, I-MC-LR and E-MC-LR of the cultures were determined as described above on day 12 of culture. Pure cultures of Maf (2 × 106 CFU ml−1) in revised HGZ media were served as the controls.
Data from co-cultures were expressed as the means ± standard deviations, and analyzed using one-way analysis of variance by the SPSS 17.0 software. Significance was set at p value of <0.05.
Nucleotide sequence accession number
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the draft genome sequence of strain JXJ CY 27-2T are MW723386 and JAKNUS000000000, respectively; the GenBank/EMBL/DDBJ accession numbers for the draft genome sequences of references strains M. invictum DSM 19600T, M. saccharophilum DSM 28107T and M. aoyamense DSM 19461T are JAKNUU000000000, JAKNUV000000000 and JAKNUT000000000, respectively.
Results and discussion
Phenotypic, physiological, and biochemical characteristics
Strain JXJ CY 27-2T was Gram-positive, aerobic and non-endospore forming. Colonies were yellow, moist, smooth and round. Cells were short rod-shaped with a size of 0.3–0.5 × 0.5–1.3 μm (Fig. S1). Growth was observed at 10.0–44.0 °C, pH 5.0–10.0 and 0–5.0% (w/v) NaCl, with optimal growth at 32.0 °C, pH 7–8 and 0% (w/v) NaCl. It is positive for catalase, starch hydrolysis, and negative for oxidase, nitrate reduction, hydrolysis of cellulose, tweens 20, 40, and 80, H2S production. Other characteristics of strain JXJ CY 27-2T and the related type strains M. invictum DSM 19600T, M. saccharophilum DSM 28107T and M. aoyamense DSM 19461T are listed in Table 1, and the detailed characteristics of the proposed novel strain are given in the species description.
The polar lipids of strain JXJ CY 27-2T are diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), unidentified glycolipids 1 and 2 (GL1 and GL2), and unidentified lipid (L1) (Supplementary Fig. S2). The main cellular fatty acids are anteiso-C17:0 (43.4%) and anteiso-C15:0 (27.1%) (Table S1), similar to the three reference strains [34,35,36]. The major menaquinones are MK 11 and MK 12. The peptidoglycan contains lysine, aspartic acid, glycine, glutamic acid and alanine. Cell-wall sugars contain mannose, ribose, rhamnose, galactose and arabinose. Arabinose was also found in the Microbacterium lacusdiani JXJ CY 01T, isolated from Microcystis .
Molecular phylogenetic analysis
Analysis of the almost-complete 16S rRNA gene sequence (1501 bp) showed that strain JXJ CY 27-2T was clearly belonged to the genus Microbacterium. It shared 16S rRNA gene sequence similarities of 98.54–98.55% with M. invictum DSM 19600T, M. saccharophilum DSM 28107T, and M. aoyamense DSM 19461T, and less than 98.47% with other members of the genus. The strain formed a distinct clade with M. invictum DSM 19600T in the phylogenetic dendrograms generated on the basis of 16S rRNA gene sequences (Figs. 1 and S3 and S4). However, it did not form clades with high bootstrap values with other type strains of the genus Microbacterium and was phylogenomically independent (Fig. S5). It was reported that the corresponding DNA reassociation values were always lower than 70% for the 16S rRNA gene sequence similarities of below 98.5% , and 16S rRNA gene sequence similarity of 98.7% was recommended as a threshold, and only species showing 98.7% or higher 16S rRNA gene sequence similarity is selected for calculating the dDDH and ANI values to determine whether the isolate is a new species [37,38,39,40]. In this study, therefore, the phylogenomic calculations of dDDH and ANI values were performed between strains JXJ CY 27-2T and M. invictum DSM 19600T, M. saccharophilum DSM 28107T, and M. aoyamense DSM 19461T. And they were 18.4 and 74.9% for M. invictum DSM 19600T, 19.9 and 75.4% for M. saccharophilum DSM 28107T, 20.3 and 75.7% for M. aoyamense DSM 19461T, which were much lower than the proposed and generally accepted species boundary values 70% and 95–96% . In fact, we also calculated the dDDH and ANI values between strain JXJ CY 27-2T and other type strains, which genomes can be downloaded from the NCBI genome database. And the dDDH and ANI values were 17.8–20.6% and 74.6–75.5%, respectively. Therefore, it strongly indicated that strain JXJ CY 27-2T represents a novel species of the genus Microbacterium, for which the name Microbacterium kunmingensis sp. nov. is proposed.
Analysis of genomic characteristics
The genomes of strain JXJ CY 27-2T, M. invictum DSM 19600T, M. saccharophilum DSM 28107T and M. aoyamense DSM 19461T were sequenced and submitted to GenBank with the accession No. JAKNUS000000000, JAKNUU000000000, JAKNUV000000000 and JAKNUT000000000, respectively. The essential features are shown in Table 2. The draft genome of strain JXJ CY 27-2T contains 28 contigs, with a total length of 3,497,265 bp and a N50 length of 300,586 bp. It has 3489 protein-coding genes with a total size of 3,221,295 bp (average 922.3 bp per protein). Therefore, the coding ratio is 92.1%. No repeat region was found. The DNA G + C content is 69.8% calculated from the genome. The numbers of tRNA and rRNA are 50 and 3, respectively. Most of the related data of strains M. invictum DSM 19600T, M. saccharophilum DSM 28107T, and M. aoyamense DSM 19461T are different from these of strain JXJ CY 27-2T (Table 2). The gene annotations of strain JXJ CY 27-2T and three reference strains in various databases are showed in Table S2.
Strain JXJ CY 27-2T had 34 genes or gene clusters (Table S3) related to the immune system process and regulation, immune response and regulation, which probably are involved in symbiotic interaction between strain JXJ CY 27-2T and Maf. Organic substances secreted by Ma can be utilized by attached bacteria as carbon and energy for survival; meanwhile, the attached bacteria can provide Ma with available N, P, CO2 and vitamins . Available nitrogen and phosphorus in eutrophic water are the two key elements inducing cyanobacterial blooms. The gene annotation data from GO database indicated that strain JXJ CY 27-2T had many genes or gene clusters of involving in phosphatases (Table S4), which can catalyze diverse phosphoric compounds to release available phosphate providing for the growth of Maf in the environments of lacking soluble phosphorus. Strain JXJ CY 27-2T also had many genes or gene clusters (including nodulation and nitrogen fixation genes) (Table S4) of involving in enzymes, which can release NH3 from many compounds or convert N2 into NH3. Therefore, strain JXJ CY 27-2T can potentially provide Maf with available N in environment of lacking available N.
Abilities of nitrogen-fixing and dissolving insoluble phosphate
The cell density increased initially from 6 × 106 CFU ml−1 to 6.6 × 108 CFU ml−1 after 4 days of culture in nitrogen-free medium, indicating that strain JXJ CY 27-2T has nitrogen-fixing ability. The contents of available phosphate increased by 0.43 ± 0.11 mg l−1 (p < 0.01) for Ca3(PO4)2 medium, and 2.29 ± 0.11 mg l−1 (p < 0.01) for phytin medium on day 2 of culture, indicating that strain JXJ CY 27-2T can dissolve insoluble phosphate.
Influences of co-culture on the growths of Maf and strain JXJ CY 27-2T
Chl-a contents of the control increased from initial 0.095 mg l−1 to 0.354, 0.530 and 0.617 mg l−1 on day 5, 10 and 15 of cultures, respectively. Different inoculation dosages of strain JXJ CY 27-2T showed different influences on the growth of Maf (Fig. 2). The chl-a contents of Maf increased to 0.368, 0.563 and 0.631 mg l−1 on day 5, 10 and 15 of cultures respectively after inoculated with strain JXJ CY 27-2T at 1 × 105 CFU ml−1, 2.3–6.3% higher (p < 0.05) than these of the controls; and the chl-a contents of Maf increased to 0.392, 0.598 and 0.646 mg l−1 on day 5, 10 and 15 of cultures respectively after inoculated with strain JXJ CY 27-2 T at 1 × 106 CFU ml−1, 4.8–12.8% higher (p < 0.01) than these of the controls (Fig. 2). The results indicated that appropriate inoculation dosages of strain JXJ CY 27-2T could promote the growth of Maf by providing with other substances except available P and N. Indole-3-acetic acid secreted by Azospirillum spp. , vitamins [6,7,8] and unknown substances  secreted by attached bacteria could promoted the cyanobacteria growth. Microcystis requires vitamin B12 for the methionine biosynthesis pathway, and other vitamins for growth . The data (Table S5) from the GO database showed that strain JXJ CY 27-2T could potentially also secrete plant growth hormones, such as indole-3-acetic acid, auxin, and polyamine, and various water-soluble vitamins, such as vitamin B1, B2, B6, B12, and K, which may be the growth-promoting factors of Maf. However, after inoculated with strain JXJ CY 27-2T at 1 × 107 CFU ml−1, the chl-a contents of Maf increased to 0.327 mg l−1 on day 5 of culture, 7.8% lower (p < 0.01) than that of the control (Fig. 2), and then increased to 0.524 and 0.607 mg l−1 on day 10 and 15 of cultures, similar to these of the controls (p > 0.05). Therefore, high inoculation dosages of strain JXJ CY 27-2T could temporarily inhibit the growth of Maf. Similarly, other attached bacteria, such as Pseudomonas sp. X , Modestobacter lacusdianchii JXJ CY 19T , and Citricoccus lacusdiani JXJ CY 21T , could also inhibit the growth of Ma under high bacterial inoculation dosages in co-cultures. High cell densities of attached bacteria probably compete with Ma for nutrients, which may further inhibit the growth of Ma temporarily.
The growth of strain JXJ CY 27-2T was inhibited in the co-cultures (Table 3). And its cell densities decreased by 22.7–75.2%, 51.7–68.3% and 42.7–91.7% in the test groups of three inoculation dosages during the test time, respectively. Similar phenomena were also found in the co-cultures of other attached bacteria and Ma [13, 42]. HGZ medium is short of organic carbon source and energy. Therefore, the organic secreta by Maf was the only sources of organic carbon and energy, which probably could not meet the growth of bacteria, and further resulted in the decrease of bacterial cells. Moreover, microcystins (MCs) may play an important ecological role, and can inhibit the growth of some bacteria , which is probably another reason for the decrease of bacterial cells. Contrary to higher inoculation dosage of strain JXJ CY 27-2T, moderate and lower inoculation dosages (1 × 105 and 1 × 106 CFU ml−1) of strain JXJ CY 27-2T promoted the growth (p < 0.05, p < 0.01) of Maf (Fig. 2), which was probably the partial reasons of a similar lower decrease rates of strain JXJ CY 27-2T in co-cultures.
Influences of co-culture on contents of MC-LR
Data in Fig. 3a showed that the E-MC-LR contents were affected by both inoculation dosages of strain JXJ CY 27-2T and culture times. The E-MC-LR content of inoculated with strain JXJ CY 27-2T at 1 × 105 CFU ml−1 was 53.4 µg mg−1 chl-a on day 5 of culture, similar to that of the control (p > 0.05); while these of inoculated with strain JXJ CY 27-2T at 1 × 106 and 1 × 107 CFU ml−1 were 46.1 and 60.1 µg mg−1 chl-a on day 5 of culture, 10.1% lower and 17.2% higher than that of the control (p < 0.05, p < 0.01), respectively. Then the E-MC-LR contents of three inoculation dosages decreased to 33.5–38.4 µg mg−1 chl-a on day 10 of culture and increased to 67.9–70.0 µg mg−1 chl-a on day 15 of culture, which were 9.4–21.0% and 29.1–31.2% lower than these of the controls (p < 0.05, p < 0.01), respectively. Therefore, in most cases, inoculation of strain JXJ CY 27-2T could reduce the release of MCs except that high inoculation dosages (≥107 CFU ml−1) could promote the release of MCs temporarily. The I-MC-LR contents were 340.5–379.9 µg mg−1 chl-a on day 10 of co-culture with strain JXJ CY 27-2T at three inoculation dosages, 11.7–20.8% lower than that of the control (p < 0.05, p < 0.01; Fig. 3b). However, the I-MC-LR contents of the groups inoculated with strain JXJ CY 27-2T showed no significant differences (p > 0.05) with that of the control on day 5 and 15 of culture.
Influences of no available N and P on Maf and JXJ CY 27-2T
In Ca3(PO4)2 medium, chl-a content of Maf co-cultured with strain JXJ CY 27-2T was 0.195 mg l−1 (Table 4) on day 12 of culture, 29.4% higher than that of the control (p < 0.01). Similar to other attached bacteria [8, 13, 14, 42], therefore, strain JXJ CY 27-2T may dissolve insoluble tricalcium phosphate to provide Maf with available phosphate for growth, which can be further proved by the genomic characteristics and genes annotated by the GO database (Table S4). In nitrogen-free medium, chl-a content of Maf co-cultured with strain JXJ CY 27-2T was 0.303 mg l−1 (Table 4), 77.2% higher than that of the control (p < 0.01). Therefore, strain JXJ CY 27-2T can convert N2 into NH3, which may be further used by Maf for growth. The ability of biological nitrogen fixation of strain JXJ CY 27-2T is consistent with the genomic characteristics and genes annotated by the GO database (Table S4).
After inoculated with strain JXJ CY 27-2T, both E-MC-LR and I-MC-LR contents of Maf were significantly affected (p < 0.01) in absence of available N and P (Table 4). In Ca3(PO4)2 medium, the contents of E-MC-LR and I-MC-LR were 222.2 and 659.8 μg mg−1 chl-a in co-culture group, which were 18.2% and 39.9% lower (p < 0.01) than these of the control group, respectively. In nitrogen-free medium, the contents of E-MC-LR and I-MC-LR were 80.3 and 410.1 μg mg−1 chl-a in co-culture group, which were 46.1% and 34.7% lower (p < 0.01) than these of the control group, respectively. Therefore, the synthesis and release of MCs of Maf were significantly inhibited by strain JXJ CY 27-2T in absence of available N and P.
Description of Microbacterium kunmingensis sp. nov
Microbacterium kunmingensis sp. nov. (kun.ming’en.sis. N.L. adj. kunmingensis. pertaining to Kunming lake, southwest China, from where the strain was isolated).
Cells are Gram-positive, aerobic, asporogenous, non-motile and short rod-shaped (0.3–0.5 × 0.5–1.3 μm). Growth occurs at 10.0–44.0 °C (optimum 32.0 °C), at pH 5.0–10.0 (optimum pH 7–8) and in the presence of 0–5.0% (w/v) NaCl. Positive for catalase and hydrolysis of starch but negative for oxidase, nitrate reduction, H2S production and hydrolysis of Tweens (20, 40, 80). Cell-wall peptidoglycan contains lysine, aspartic acid, glutamic acid, glycine and alanine. Cell-wall sugars contain mannose, ribose, rhamnose, galactose and arabinose. Polar lipids consist of DPG, PG, unidentified glycolipid 1, unidentified glycolipid 2, and an unidentified lipid. Predominant cellular fatty acids are anteiso-C17:0, anteiso-C15:0, iso-C17:0, iso-C16:0, and iso-C15:0. Major menaquinones are MK 11 and MK 12. The DNA G + C content based on the draft genome sequence is 69.8%.
The type strain, JXJ CY 27-2T (=CGMCC 1.17506T = KCTC 49382T), was isolated from the culture of Microcystis aeruginosa FACHB-905, which was collected from Lake Kunming, Yunnan province, south-west China. The GenBank accession numbers for the 16S rRNA gene sequence and draft genome sequence of strain JXJ CY 27-2T are MW723386 and JAKNUS000000000, respectively.
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This research was supported by funds from Natural Science Foundation of China (No. 31060010).
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The authors declare no competing interests.
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Xiao, Y., Chen, M., Chen, J. et al. Microbacterium kunmingensis sp. nov., an attached bacterium of Microcystis aeruginosa. J Antibiot 75, 662–670 (2022). https://doi.org/10.1038/s41429-022-00568-w