Modestobacter lacusdianchii sp. nov., a Phosphate-Solubilizing Actinobacterium with Ability to Promote Microcystis Growth.

A novel actinobacterium, designated strain JXJ CY 19T, was isolated from a culture mat of Microcystis aeruginosa FACHB-905 collected from Dianchi Lake, South-west China. 16S rRNA gene sequences comparison of strain JXJ CY 19T and the available sequences in the GenBank database showed that the strain was closely related to Modestobacter marinus 42H12-1T (99.1% similarity) and Modestobacter roseus KLBMP 1279T (99.0%). The isolate had meso-diaminopimelic in the cell wall with whole-cell sugars of mannose, rhamnose, ribose, glucose, galactose, and arabinose. The menaquinone detected was MK-9(H4), while the major cellular fatty acids include C17:1 ω8c, C15:0 iso, C15:1 iso G and C16:0 iso. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and an unidentified phospholipid. The DNA-DNA hybridization values between strains JXJ CY 19T and the closely related type strains Modestobacter marinus CGMCC 4.5581T and Modestobacter roseus NBRC 108673T were determined to be 50.8 ± 0.8% and 44.1 ± 1.7%, respectively. The DNA G+C content was 71.9 mol%. On the basis of the above taxonomic data and differences in physiological characters from the closely related type strains, strain JXJ CY 19T was recognized as a novel species of the genus Modestobacter, for which the name Modestobacter lacusdianchii sp. nov. (JXJ CY 19T = KCTC 39600T = CPCC 204352T) is proposed. The type strain JXJ CY 19T can solubilize calcium phosphate tribasic (Ca3(PO4)2), phytin and L-α-phosphatidylcholine. The phosphate-solubilizing property of the novel actinobacterium could be a possible factor for the increase in growth of Microcystis aeruginosa FACHB-905 in ecosystem where the amount of available soluble phosphate is limited such as Dianchi Lake.

Introduction

Dianchi Lake, the largest freshwater lake in Yunnan Province and the sixth largest in China, has been heavily polluted owing to unchecked inflow of industrial, agricultural, and domestic wastes. The untreated disposal of waste is the leading cause for high biochemical oxygen demand, nitrate and phosphate, thereby providing a source for increase of cyanobacterial blooms [1]. The algal blooms which predominantly occur in warm season are dominated by the genus Microcystis [1], of which the most common one is that of Microcystis aeruginosa [2, 3]. The Microcystis mat is found to be associated with a host of other bacteria including those belonging to the phyla Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria and Bacteroidetes [4-7]. Co-existence between the two types of microorganisms is apparently maintained by the supply of growth factors, microelement [4], phosphate [8] and probably available carbon source (CO2) [9] by the bacteria to Microcystis, which in turn, provide organic nutrients [4, 10–12] and safer growing environment [4, 13] to the bacteria.

During analysis of culture mat of Microcystis aeruginosa FACHB-905 isolated from Dianchi Lake (http://algae.ihb.ac.cn/), a phosphate-solubilizing novel actinobacterium designated strain JXJ CY 19T belonging to the genus Modestobacter was isolated. This manuscript described the polyphasic characterization of this actinobacterial strain. The manuscript also reports the effect on the growth of M. aeruginosa under in vitro conditions by co-culturing with the novel actinobacterial strain JXJ CY 19T.

Materials and Methods

Isolation and maintenance of strain

About 0.2 ml of M. aeruginosa FACHB-905 culture obtained from Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB collection), Chinese Academy of Sciences (CAS), Wuhan, China was spread on International Streptomyces Project medium 2 (ISP 2) [14] and incubated at 28°C for 5 days. Bacterial colonies arising on the isolation media were selected and repeatedly streaked on ISP 2 agar plates to obtain pure cultures. Purified strain JXJ CY 19T was maintained on ISP 2 slants at 28°C and stored as glycerol suspensions (30%, v/v) at -80°C.

Phenotypic characteristics

Morphology was observed using light microscope (BX43; Olympus) and electron microscope (QUANTA200; FEI). Gram staining was carried out by using the standard Gram’s stain procedure. Growth at various temperatures (4–50°C), pH (4.0–11.0) and NaCl concentrations (0–10%, w/v) were examined according to method described by Xu et al. [15] using ISP 2 as the basal medium. Catalase activity was determined using H2O2 (3%). Oxidase activity was tested according to Kovacs [16]. Other phenotypic characteristics were determined according to Goodfellow [17] and Williams et al. [18]. Enzyme activities were tested by using the commercial API ZYM system (bioMérieux). Assimilation of various substrates was tested using Biolog GN III Micro Plate assays following manufacturer’s instructions.

Chemotaxonomy

Analysis of isomer of diaminopimelic acid and whole-cell sugars were performed according to the procedures developed by Hasegawa et al. [19] and Tang et al. [20] respectively. Polar lipids were extracted according to the method described by Minnikin et al. [21] and analyzed as described by Collins & Jones [22]. Menaquinones were extracted according to the method described by Collins et al. [23] and analyzed using HPLC [24]. Analysis of fatty acids was performed by GC using the microbial identification system (Sherlock Version 6.1; MIDI database: TSBA6) [25]. Biomass for fatty acid analysis was obtained from cells grown on tryptone soy agar (TSA; Difco) at 28°C for 4 days. The G+C content of genomic DNA of strain JXJ CY 01T was determined by using HPLC [26].

Molecular analysis

16S rRNA gene sequence of strain JXJ CY 19T was aligned with sequences of the most closely related taxa by using CLUSTAL_X program version 1.83 [27]. Phylogenetic trees were constructed by using the neighbour-joining [28], maximum-likelihood [29] and maximum-parsimony [30] tree-making algorithms using MEGA version 5.0 software [31]. Topologies of the phylogenetic trees were evaluated by bootstrap analysis of Felsenstein [32] with 1000 replicates. The genomic relatedness between strain JXJ CY 19T and closely related strains were performed as described by Ezaki et al. [33].

Phosphate solubilization

The ability of strain JXJ CY 19T and other members of the genus Modestobacter to solubilize insoluble phosphate were determined on plates using phosphate-solubilizing media [glucose, 10 g; (NH4)2SO4, 0.5 g; MgSO4·7H2O, 0.3 g; NaCl, 0.3 g; KCl, 0.3 g; FeSO4·4H2O, 0.036 g; MnSO4·4H2O, 0.03 g; Ca3(PO4)2, 10 g or L-α-phosphatidylcholine, 2.0 g or phytin, 2.0 g; distilled water, 1000 ml; pH 7.0] [34]. Since some microorganisms can solubilize insoluble phosphates in liquid medium despite showing no clear phosphate-solubilizing zone on agar plates [35], the phosphate-solubilizing ability is further confirmed using liquid cultures. Cultures of the tested strains grown in ISP 2 broth (28°C, 2–5 days) were centrifuged (4,860×g, 20 min, 4°C), and the biomass resuspended in small aliquots of sterilized distilled water. Cell suspension was inoculated into the phosphate-solubilizing media with a final cell density of 1×106 CFU/ml. For the control, the bacterial cell inoculum was replaced with sterile water. Culture broth was centrifuged (4,860×g, 20 min) on the 7th day of incubation, and the amount of available phosphorus in the supernatant (measured as phosphate equivalent) determined colorimetrically using standard protocol as described below [34].

Reaction mixtures containing 5 ml supernatant, 0.1 ml 2,4-dinitrophenol solution (0.011 M) and 5 ml Mo-Sb reagent solution were adjusted to a final volume of 50 ml with distilled water, briefly mixed and kept incubated at 20°C for 30 min. Absorbance of the reaction mixture was monitored at 700 nm, and the available phosphorus in each reaction mixture determined against a standard curve of potassium phosphate. The Mo-Sb reagent solution contained (per liter) sulfuric acid, 2.87 mol; ammonium molybdate, 8.1 mmol; antimonyl potassium tartrate, 1.5 mmol; ascorbic acid, 85.2 mmol (added into the solution just before use).

Concentration of the available phosphorus in the culture media was calculated based on the following equation:

where X represent available phosphorus in the culture media,

P, available phosphorus in the reaction mixture,

V1, total volume of reaction mixture,

V2, volume of culture supernatant added in the reaction mixture, and

K, the dilution ratio used for measuring the absorbance.

Effect on the growth of M. aeruginosa by co-culturing with strain JXJ CY 19T under in vitro condition

As M. aeruginosa FACHB-905 culture mat has many associated bacteria, the culture FACHB-905 is purified prior to co-culture for understanding the effects of strain JXJ CY 19T on its growth. 0.1 ml M. aeruginosa FACHB-905 culture mat was spread on HGZ agar medium [36] and kept incubated under illumination of 30–50 μmol photon/m2/s on a 12-h light/dark cycle at 25°C. The cultures were incubated until green colonies were observed. These colonies were checked for bacterial contamination by spreading on ISP 2 agar, with parallel observation under light microscope. Absence of bacterial growth on ISP 2 indicated a pure M. aeruginosa culture preparation. Pure colonies were further inoculated into fresh HGZ media, and incubated for another 30 days.

Biomass of purified M. aeruginosa FACHB-905 were collected by centrifugation (4,860×g, 20 min, 4°C) and inoculated into HGZ and modified HGZ media (KH2PO4 replaced with Ca3(PO4)2 or L-α-phosphatidylcholine) with an initial density of approximately 2×106 CFU/ml. The media were then co-inoculated with strain JXJ CY 19T corresponding to inoculum densities of 0.2×107 CFU/ml, 1×107 CFU/ml and 5×107 CFU/ml. For the control, bacterial cell suspension was replaced by sterilized water. The co-cultures were kept incubated under illumination of 30–50 μmol photon/m2/s in a 12-h light/dark cycle. Cells of M. aeruginosa were counted under light microscope (Olympus BX43, Japan) on the 7th and 60th days of incubation and while for strain JXJ CY 19T, plate colony counting method was adopted to determine the CFU. The available phosphorus in the modified HGZ media was also concurrently measured.

Statistical analysis

All the experiments for phosphate solubilization and determination of CFU were done in triplicates, and the values were expressed as their mean. These data were subjected to one-way ANOVA at P < 0.05 and P < 0.01 using SPSS 17 software (SPSS Inc).

Results

Strain JXJ CY 19T was Gram-stain positive and non-endospore-forming. Cells of strain JXJ CY 19T were short rods (straight or lightly curved) with size of 0.5–1 × 1.0–2.5 μm when cultivated in ISP 2 broth for less than 24 hours. The cells gradually turned coccoid in the later stages. Strain JXJ CY 19T could grow at 4–40°C, pH 6.0–9.0 and 0–6% (w/v) NaCl, with optimal growth at 25–28°C, pH 7.0–8.0 and 0–3% (w/v) NaCl. The isolate was found positive for catalase and oxidase tests.

Detailed phenotypic characteristics of the strain are given in Table 1 and species description.

Table 1

Comparative characteristics between strain JXJ CY 19T and other members of the genus Modestobacter.

	1	2	3	4[37]*	5[38]*	6[38]*	7[39]*
Colony colour on ISP 2 medium	Pink	Pink-deep orange to dark	Pink	Skin-colour to pale pink	Light-dark orange	Light orange-pink	Pink-deep orange to dark
G+C content (mol %)	71.9	72.3*	71.7*	69.9	72	71.5	73
Major fatty acids (>10%)	C15:0 iso, C16:0 iso, C15:1 iso G, C17:1 ω9c	C16:0 iso, C16:0*, C17:1 ω9c*, C17:0*	C16:0 iso, C17:1 ω9c, C15:0 iso*	C15:0 iso, C16:0 iso	C16:0 iso, C15:0 iso, C16:1 ω9c	C17:1 ω9c, C17:0, C16:0 iso	C16:0 iso, C15:0 iso
Polar lipids	DPG, PE, PI, PIM, PL	DPG, PE, PG, PI, APL*	DPG, PE, PI, PIM, APL, PL*	DPG, PE, PI	DPG, PE, PG, PI, PIM	DPG, PE, PG, PI, PIM	DPG, PG, PE, PI
Whole-cell sugars	Ara, Gal, Glu, Man, Rha, Rib	ND	Gal, Glu, Rib*	Gal, Glu, Rib	Ara, Gal, Glu, Rib	Gal, Glu, Rib	Gal, Glu, Rib
H2S	-	+	-	-	ND	ND	-
NaCl range (w/v, %)	0–6	0–6	0–9	0–8	0–8	0–8	0–3
Growth temperature range (°C)	4–40	4–40	10–40	0–28	20–37	20–37	4–30
pH range for growth	6–9	7–9	6–9	3–12	6–9	5–9	5–9
Nitrate reduction	-	+	-	+	+	+	+
Urease activity	-	-	-	ND	+	+	+
Utilization of
D-Arabinose	-	+	+	ND	ND	ND	-
D-(+)-Cellobiose	+	-	+	-	+	-	+
D-Glycerol	+	-	w	+	-	-	+
Maltose	+	+	+	w	+	-	+
Lactose	-	-	+	+	-	-	+
Myo-inositol	-	-	-	-	-	-	+
D-Galactose	+	+	+	+	-	-	-
Mannitol	+	+	+	+	ND	ND	+
Salicin	-	-	+	+	-	-	+
D-Mannose	+	+	-	w	-	-	+
L-Rhamnose	+	+	w	w	-	-	+
D-Sorbitol	+	+	w	-	+	-	-
D-Melibiose	+	-	+	-	+	-	+
Sodium acetate	+	-	-	+	ND	ND	-
Citric acid	+	-	-	-	+	-	-
Malate	-	+	+	+	+	+	+
L-Alanine	+	+	-	-	-	-	+
L-Glycine	+	-	-	ND	ND	ND	-
L-Phenylalanine	+	+	-	ND	ND	ND	-
Dextrin	+	-	w	-	+	+	-
D-Fucose	-	+	w	+	-	-	+
Inosine	+	-	w	+	+	-	+

1, JXJ CY 19; 2, M. marinus CGMCC 4.5581T; 3, M. roseus NBRC 108673T; 4, M. multiseptatus AA-826T; 5, M. lapidis MON 3.1T; 6, M. muralis MDVD1T; 7, M. versicolor DSM 16678T.

+: positive; -: negative; w: weakly; ND: no determined.

Data for strains JXJ CY 19, M. marinus CGMCC 4.5581T, M. roseus NBRC 108673T, except those marked by asterisks, are from this study.

DPG: diphosphatidylglycerol; PG: phosphatidylglycerol; PE: phosphatidylethanolamine; PI: phosphatidylinositol; PIM: phosphatidylinositol mannosides; APL, unidentified aminophospholipid(s); PL, unidentified phospholipid(s); L, unidentified lipid(s).

Ara: arabinose; Gal: galactose; Glu: glucose; Man: mannose; Rha: rhamnose; Rib: ribose.

Strain JXJ CY 19T contained meso-DAP, along with mannose, rhamnose, ribose, glucose, galactose, and arabinose in the whole-cell hydrolysates. Polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and an unidentified phospholipid (S1 Fig). The menaquinone detected was MK-9 (H4). The fatty acids profile was C15:0 iso (25.4%), C16:0 iso (25.3%), C15:1 iso G (10.2%), C17:1 ω9c (9.9%), summed feature 3 comprising C16:1 ω6c and/or C16:1 ω7c (4.4%), C17:0 iso (3.7%), summed feature 9 comprising C16:0 10-methyl (3.1%), C16:0 (3.0%), C16:1 iso H (2.6%), C17:0 (1.4%) and C15:1 ω6c (1.2%). The G+C content of the genomic DNA was determined to be 71.9 mol%.

Strain JXJ CY 19T showed highest 16S rRNA gene sequence similarities with members of the genus Modestobacter: Modestobacter marinus 42H12-1T, 99.12%; M. roseus KLBMP 1279T, 99.03%; M. versicolor CP 153-2T, 98.41%; M. muralis MDVD1T, 98.21%; M. lapidis MON 3.1T, 97.99% and M. multiseptatus AA-826T, 97.64%. The strain formed a stable clade with strains M. marinus 42H12-1T and M. roseus KLBMP 1279T in the phylogenetic dendrograms based on 16S rRNA gene sequences (Fig 1; S2 and S3 Figs), indicating that the strain belongs to the genus Modestobacter. Based on the analysis of the 16S rRNA gene sequences, the phylogenetic trees and the recommendation of Stackebrandt and Ebers [40], the two strains M. marinus CGMCC 4.5581T and M. roseus NBRC 108673T were considered for DNA-DNA relatedness study. The DNA-DNA hybridization values between strain JXJ CY 19T and type strains M. marinus CGMCC 4.5581T and M. roseus NBRC 108673T were determined to be 50.81 ± 0.84% and 44.07 ± 1.66% respectively (S1 Table).

Fig 1

Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences of strain JXJ CY 19T and representative type strains of the family Geodermatophilaceae.

Bootstrap values (expressed as percentages of 1,000 replications) of above 50% are shown at the nodes. Asterisks indicate clades that were conserved using the maximum-parsimony and maximum-likelihood methods. Bar, 0.005 sequence divergence.

In addition to the differences in the genomic DNA relatedness, strain JXJ CY 19T could be differentiated from the type strains of the genus Modestobacter by the characteristics listed in Table 1. Based on the data in this study, we propose that strain JXJ CY 19T represents a novel species of the genus Modestobacter, for which the name Modestobacter lacusdianchii sp. nov. is proposed.

Description of Modestobacter lacusdianchii sp. nov.

Modestobacter lacusdianchii sp. nov. (la.cus.di.a'n.chii L. gen. n. lacus, of a lake; N.L. gen. n. dianchii, of Dianchi; N.L. gen. n. lacusdianchii, of Dianchi lake).

Cells are aerobic, Gram-staining positive, non-spore-forming, short rods (0.5–1.0 × 1.0–2.5 μm, straight or lightly curved), or cocci with a tendency to aggregate. Colonies are pink throughout growth. Growth is observed at 4–40°C, pH 6.0–9.0 and 0–6% (w/v) NaCl, with optimal growth at 25–28°C, pH 7.0–8.0 and 0–3% (w/v) NaCl. Utilizes D-(+)-cellobiose, D-fructose, D-galactose, D-glucose, D-glycerol, myo-inositol, D-mannitose, D-mannose, D-raffinose, D-sorbitol, L-rhamnose, sucrose, D-trehalose, D-xylose, D-melibiose, sodium acetate, L-lactose and dulcitol as sole carbon sources, but not D-arabinose, D-ribose, D-xylitol or sodium propionate. Utilizes L-alanine, L-arginine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-leucine, L-isoleucine, L-methionine, L-lysine, L-phenylalanine, L-valine, L-threonine, L-tyrosine, L-proline, L-tryptophan, L-serine and hypoxanthine as sole nitrogen sources, but not L-histidine. Positive for catalase, oxidase and phosphatase assays, but negative for milk coagulation, milk peptonization, nitrate reduction, methyl red test, Voges-Prokauer test, H2S production, and hydrolysis of casein, gelatin, cellulose and Tweens 20, 40, 60 and 80. Acid is produced from starch, aesculin and D-tagatose (API 50 CH). Cell-wall peptidoglycan contains meso-DAP, with mannose, rhamnose, ribose, glucose, galactose, and arabinose as whole-cell sugars. Polar lipids consist of phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides. The menaquinone is MK-9(H4). Major fatty acids are C17:1 ω8c, C15:0 iso, C15:1 iso G and C16:0 iso. The G+C content of the genomic DNA of the type strain is determined to be 71.9 mol%.

The type strain, JXJ CY 19T (= KCTC 39600T = CPCC 204352T), was isolated from the culture mat of Microcystis aeruginosa FACHB-905 collected from Dianchi Lake, China. The 16S rRNA gene sequence of strain JXJ CY 19T has been deposited in GenBank under the accession number KP986567.

All the seven tested strains formed no visible halo zones for solubilization of either Ca3(PO4)2 or L-α-phosphatidylcholine in plates, while strains M. marinus CGMCC 4.5581T, M. roseus NBRC 108673T, M. versicolor CP 153-2T and M. muralis MDVD1T formed weak zone for solubilization of phytin. Under liquid culture assay, all the strains could solubilize L-α-phosphatidylcholine and phytin with a detection of additional available phosphorus of 0.5–0.9 mg/l and 0.5–4.5 mg/l respectively than the control. Only strain JXJ CY 19T and M. muralis MDVD1T were able to solubilize Ca3(PO4)2 with an available phosphorus content of 0.2–0.3 mg/l higher than that of the control

Effect on the growth of M. aeruginosa FACHB-905 by co-culturing with strain JXJ CY 19T under in vitro condition

Cell density of M. aeruginosa FACHB-905 in the control media increased from initial 2×106 CFU/ml at day 0 to 9.03×106 CFU/ml and 9.87×107 CFU/ml on day 7 and 60 respectively in the absence of bacterial co-inoculant. Co-culturing with 0.2×107 CFU/ml of JXJ CY 19T enhanced the cell density of M. aeruginosa from the control by 10.29% and 12.06% (P < 0.05) on day 7 and day 60 respectively (Tables 2 and 3). However, with increased bacterial inoculum density (5×107 CFU/ml), cyanobacterial cell growth were initially observed to decrease on day 7 but recovered significantly on day 60, as compared to control at P < 0.01 (Tables 2 and 3).

Table 2

Cell numbers (mean±standard deviation; n = 3) of Microcystis aeruginosa FACHB-905 after co-culturing with strain JXJ CY 19T for 7 days.

Cell density of strain JXJ CY 19T (×107 CFU/ml)		Cell density of FACHB-905 (×106 CFU/ml)
Initial	Final	HGZ media	a	b
0	0	9.03±0.33	6.85±0.31	6.72±0.42
0.2	~0.1	9.96±0.45*	7.45±0.36	8.90±0.38**
1.0	~0.3	8.70±0.39	8.13±0.51*	9.15±0.49**
5.0	~1.0	7.75±0.24**	9.15±0.41**	9.60±0.44**

Statistical comparisons with the controls were made using ANOVA (* P < 0.05, ** P < 0.01).

a and b represented KH2PO4 in HGZ media was replaced with Ca3(PO4)2 and L-α-phosphatidylcholine, respectively.

Table 3

Cell numbers (mean±standard deviation; n = 3) of Microcystis aeruginosa FACHB-905 after co-culturing with strain JXJ CY 19T for 60 days.

Cell density of strain JXJ CY 19T (×107 CFU/ml)		Cell density of FACHB-905 (×106 CFU/ml)
Initial	Final	HGZ media	a	b
0	0	9.87±0.32	5.58±0.22	6.38±0.37**
0.2	~0.03	11.06±0.44*	11.36±0.58**	10.21±0.46**
1.0	~0.06	12.26±0.62**	17.56±0.90**	11.95±0.54**
5.0	~0.30	18.53±0.88**	18.03±0.92**	12.41±0.40**

Statistical comparisons with the controls were made using ANOVA (* P < 0.05, ** P < 0.01).

a and b represented KH2PO4 in HGZ media was replaced with Ca3(PO4)2 and L-α-phosphatidylcholine, respectively.

When KH2PO4 in the medium is replaced either by Ca3(PO4)2 or L-α-phosphatidylcholine, the increase in cyanobacterial cell density is quite significant (P < 0.05, P < 0.01), and become more prominent with increase in inoculum density of strain JXJ CY 19T (Tables 2 and 3). This increase may be accounted for by the solubilization of the insoluble phosphate (Fig 2; P < 0.01).

Fig 2

Available phosphorus concentrations of different co-cultures of in modified HGZ media on day 7 (A) and 60 (B). a, b, c, and d represented the treatments of initial bacterial cell densities of 0×107 CFU/ml, 0.2×107 CFU/ml, 1×107 CFU/ml and 5×107 CFU/ml respectively. Statistical comparisons with the control were made using ANOVA (* P < 0.05, ** P < 0.01).

Discussion

Many studies have been conducted to understand the phytoplankton communities of eutrophic lakes [1, 4]. It was usually reported that algae especially Microcystis dominate this communities [1-4]. In a similar study by Parveen et al. [7], Microcystis colonies appeared to be depleted of Actinobacteria, while enriched in Gammaproteobacteria. In our earlier studies [36, 41], we have found the presence of antialgal compounds from a novel Streptomyces jiujiangenesis strain JXJ 0074T [42]. This might be another reason for a non-cohesive existence of free living Actinobacteria in the phytoplankton communities of eutrophic lakes as reported by Parveen et al. [7]. In contrast to the above studies, the present study indicates the presence of a growth-promoting actinobacteria of the genus Modestobacer within these lake bacterial communities.

The genus Modestobacter belongs to the family Geodermatophilaceae [43, 44] of the order Geodermatophilales [45]. Like other strains of the order Geodermatophiles, the genus Modestobacter tend to be associated with extreme biomes, including deteriorated sandstone [38], desert plateau [39], deep-sea sediment [46] and coastal halophytes [47]. The present study reports the isolation of a Modestobacter strain from a eutrophic lake located in Yunnan, China. Interestingly all the Modestobacter strains were found to solubilize insoluble phosphorus despite differences in their origins.

Phosphorus is a key chemical element essential for biological activities. Only 5–8% of the total phosphorus in the water was available to biology [48] and is, therefore, considered as the principal limiting nutrient for algal growth in most freshwater habitats [49, 50]. Microcystis mat are often found associated with many bacteria in a complex relationship. Among these bacteria isolated from different freshwater cyanobacterial mat, the strains Pseudomonas sp. X, Erythrobacter sp. Y6, Gordonia sp. txj1302RI and Burkholderia sp. txj1302Y4 have been found to decompose the insoluble phosphate and thereby making it available for the growth of Microcystis [8, 51–53]. Similar result is found during the present study The novel strain JXJ CY 19T was found to solubilize inorganic and organic phosphate from insoluble source (Fig 2) under in vitro condition, and this soluble phosphorus are made available for growth of M. aeruginosa FACHB-905 (Tables 2 and 3).

In addition to the availability of phosphorus source, additional factors might also be responsible for the increase in growth of cyanobacteria. This is indicated by the fact that despite adequate phosphorus in the normal HGZ media (Fig 2), co-culturing with lower cell density of strain JXJ CY 19T result in significant increase in growth of M. aeruginosa (Tables 2 and 3). Similar findings have been reported by several studies of cyanobacterial mat-associated bacteria. Zhao et al. [8] found that Actinobacteria strain Gordonia sp. txj1302RI produce unknown substances that promote the growth of Microcystis. de-Bashan et al. [54] reported that Azospirillum spp. produce indole-3-acetic acid that helps in promoting the growth of Chlorella vulgaris. The symbiotic relationship of M. aeruginosa is also influenced by the cellular density of the associated bacteria. Under condition of abundant phosphorus, lower cell density (< 0.2×107 CFU/ml) of strain JXJ CY 19T stimulate the growth of M. aeruginosa FACHB-905 while and higher cell density (~1–5×107 CFU/ml) inhibit its growth (Table 2). With time, the cell densities of M. aeruginosa recuperate, but with a concomitant decrease in the cell density of strain JXJ CY 19T.

Two-dimensional thin-layer chromatogram of polar lipids of strain JXJ CY 19T stained with 5% ethanolic molybdophosphoric acid.

The chromatographic conditions were as follows: Silica Gel 60 thin-layer plates (10 by 10 cm) were spotted with 10 μl of a whole-cell lipid extract. Chloroform-methanol-water (65:25:4, v/v/v) was used to develop the chromatogram in the first direction, and chloroform-acetic acid-methanol-water (80:18:12:5, v/v/v/v) was used in the second direction. DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIM, phosphatidylinositol mannosides; PL, unidentified phospholipid.

(PDF)

Click here for additional data file.

Maximum-Parsimony phylogenetic tree based on 16S rRNA gene sequences of strain JXJ CY 19T and representative type strains of the family Geodermatophilaceae.

Bootstrap values (expressed as percentages of 1000 replications) of above 50% are shown at the branch points.

(PDF)

Click here for additional data file.

Maximum-Likelihood phylogenetic tree based on 16S rRNA gene sequences of strain XJ CY 19T and representative type strains of the family Geodermatophilaceae.

Bootstrap values (expressed as percentages of 1000 replications) of above 50% are shown at the branch points. Bar, 0.005 sequence divergence.

(PDF)

Click here for additional data file.

DNA–DNA relatedness between strain JXJ CY 19T and closely related members of the genus Modestobacter.

A, JXJ CY 19T; B, M. marinus CGMCC 4.5581T; C, M. roseus NBRC 108673T.

(PDF)

Click here for additional data file.