Introduction

The family Rhodobacteraceae, which belongs to the class Alphaproteobacteria and represents a phenotypically, metabolically, ecologically diverse group of bacteria, was initially proposed by Garrity et al. [1]. The family Rhodobacteraceae contains 174 genera with validly published names at the time of writing (www.bacterio.net/rhodobacteraceae.html). Some bacteria of this family such as genera Rhodobacter and Rhodovulum are able to perform anoxygenic photosynthesis [2]. Phototrophy is recognized as being specific for certain genera of this family and is an important characteristic in differentiating the genera of phototrophs from those of chemotrophs [3, 4]. Species of genus Rhodobacter containing bacteriochlorophyll a are phototrophs. The genus Rhodobacter embrace 16 validly named species at the time of writing [5]. The species of this genus have been mostly isolated from various aquatic environments, such as hot spring sediment [5], semiarid tropical soil [6], marine habitats [7], and pond [8]. The aim of this study is to investigate the taxonomic position of a Rhodobacter-like strain using a polyphasic taxonomic approach.

Materials and Methods

Bacterial Isolation

The activated sludge was collected from an abandoned herbicide manufacturing plant (E120°56′38″, N31°22′05″) in Kunshan City, Jiangsu Province, China. The sludge sample was streaked on R2A agar. A yellowish colony, designated strain HX-7-19T, was picked and purified after incubation for 7 days at 30 °C and stored at − 70 °C in R2A broth supplemented with 20% (v/v) glycerol. Rhodobacter blasticus CGMCC 1.3365T was used as a reference strain and was cultured in R2A at 30 °C.

PCR Assay

Genomic DNA of HX-7-19T was extracted using a bacterial genomic kit according to the manufacturers’ protocols (TIANamp Bacteria DNA Kit, Tiangen). The DNA was used as a template to amplification of 16S rRNA gene and photosynthetic genes (pufL and pufM). The bacterial universal primers 27F and 1492R were used to amplify the 16S rRNA gene sequence [9]. The amplified product was purified with an agarose gel DNA extraction Kit (Shanghai Sangon biotech, China), and then ligated into pMD-18T vector (Takara Biotechnology), and sequenced by an automated sequencer (model 3730, Applied Biosystems). The 16S rRNA gene sequence was compared with known sequences found in GenBank using the BLAST program of the NCBI (www.ncbi.nlm.nih.gov/BLAST/) and also identified in EzBioCloud’s Identify service (www.ezbiocloud.net/identify). Sequence alignment was performed using the CLUSTAL_W program. Phylogenetic trees were reconstructed based on the neighbor-joining (NJ) algorithms [10], maximum-likelihood (ML) algorithms [11] and minimum-evolution (ME) algorithms [12], and were carried out by the MEGA software (version 7.0) according to Kimura’s two-parameter calculation model [13], and were assessed using bootstrap analysis of 1000 replications [14]. The pufL and pufM genes were amplified by using the primer pair pufLM-67F (5′-TTCGACTTYTGGRTNGG NCC-3′) and pufLM-781R (5′-CCAKSGTCCAGCGCCAGAANA-3′) [15]. The expected length of the amplified pufLM genes fragment is about 1.5 kb. R. blasticus CGMCC 1.3365T was used as the positive control.

Chemotaxonomic Characterization

For chemotaxonomic analysis, strain HX-7-19T and the closest phylogenetically related strain R. blasticus CGMCC 1.3365T were cultured in R2A on a rotary shaker (180 rpm) at 30 °C for 7 days and 5 days, respectively. The cells were harvested by centrifugation at exponential growth phases, and then washed with distilled water and freeze-dried. Cellular fatty acid were saponified, methylated, extracted, and analyzed according to the standard protocol of the Sherlock MIS (MIDI) system. The respiratory quinones were tested according to the method as described previously [16]. Polar lipids were extracted using a chloroform/methanol system [17, 18] and analyzed according to the method as described previously [19].

Genomic DNA of strain HX-7-19T was extracted and purified according to standard procedures, and then sequenced and assembled by Illumina Hiseq 4000 platform at Shanghai Biozeron Biotechnology Co., Ltd, China. The assembled genomes were annotated with the Rapid Annotation with Subsystem Technology (RAST) server (https://rast.nmpdr.org/). The average nucleotide identity (ANI) between strain HX-7-19T and R. blasticus CGMCC 1.3365T were calculated using a tool of OrthoANIu algorithm (www.ezbiocloud.net/tools/ani) [20]. The digital DNA–DNA hybridization (dDDH) were calculated by the genome-to-genome distance calculator (http://ggdc.dsmz.de/ggdc.php/) [21]. The DNA G + C content was determined from the genome sequence.

Morphological, Physiological, and Biochemical Characterization

Cells of strain HX-7-19T (exponential growth phase) were observed using a transmission electron microscopy (H-7650; Hitachi) to determinate the morphological characterization. The characterization of motility was observed using an optical microscope (BX40; Olympus) according to the hanging-drop method [22]. Gram staining was tested using a Gram-stain kit (Difco) according to the instructions of the manufacturer. The temperature range (4, 10, 15, 18, 20, 25, 30, 37, 42, and 47 °C) and pH range (pH 3.0–9.0 using increments of 0.5 pH units) for growth were determined by incubating the isolate for one week in R2A broth. The pH range was adjusted by using buffer system according to method by Xu et al. [5]. Growth in various NaCl concentrations (0–5%, w/v, at 0.5% interval) was evaluated in R2A broth. Growth was monitored by measuring OD600 nm by a UV-1800 spectrometer (Shimadzu Corp.; Japan).

Photo-organoheterotrophic growth of HX-7-19T was investigated in Pfennig medium containing sodium pyruvate (0.3%, w/v) and NH4Cl as the carbon and nitrogen source under light exposure (2400 lx) and anaerobic conditions at 30 °C [23]. Chemo-organoheterotrophic growth was determined in Pfennig medium containing sodium pyruvate (0.3%, w/v) as the only carbon source under dark and aerobic conditions. The chemo-lithoautotrophically growth was tested under aerobic and dark conditions with Na2S (0.5 mM), Na2S2O3 (0.5 mM) as electron donors and NaHCO3 (0.1%, w/v) as carbon source, and fermentative growth was investigated at anaerobic and dark conditions with pyruvate (0.3%, w/v) as fermentable substrate. Vitamin (vitamin B12, biotin, niacin, paminobenzoic acid, and thiamine) requirement was tested by replacing yeast extract with single and also combinations of vitamins as growth factors. Biochemical characters of strain HX-7-19T were studied using commercial identification kits (API 20NE, API 50CH, and API ZYM) and Biolog GENIII MocroPlate according to the manufacturers’ protocols (bioMérieux).

Results and Discussion

Phylogenetic and Genomic Analysis

The sequenced length of 16S rRNA gene of strain HX-7- was 1453 bp. Comparative analysis of 16S rRNA gene sequences indicated that strain HX-7-19T was closely related to those of R. blasticus CGMCC 1.3365T (96.3% sequence similarity), and were lower than 97% with other type strains of family Rhodobacteraceae. The phylogenetic tree showed that strain HX-7-19T formed a distinct clade with the related type strain R. blasticus CGMCC 1.3365T according to NJ algorithm (Fig. 1). The affiliation result was consistent with the ME algorithm (Fig. S6) and ML algorithm (Fig. S7).

Fig. 1
figure 1

Neighbor-joining (NJ) phylogenetic tree based on 16S rRNA gene sequences, showing the taxonomic position of strain HX-7-19T and related taxa. Bootstrap values were calculated from 1000 replications and values below 50% are not indicated at branch points. Bar, 0.01 substitutions per nucleotide position

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The draft genome size of strain HX-7-19 T (4.46 Mb) was much bigger than that of R. blasticus CGMCC 1.3365T (3.59 Mb). The predicted number CDSs and RNA genes of strain HX-7-19 which were higher than those determined in the genome of R. blasticus CGMCC 1.3365T. The G + C content of the genome (65.9 mol%) was a slightly smaller than that of R. blasticus CGMCC 1.3365T (66.5 mol%) (Table 1). In the subsystem features (subsystem coverage and subsystem category distribution), some differences were observed at the two genomes (Table 1). For example, the numbers of genes putatively involved in cofactors, vitamins, prosthetic groups, pigments, cell wall and capsule, and metabolisms of RNA, DNA, phosphorus, sulfur, and aromatic compounds in genome of HX-7-19T were higher than those of R. blasticus CGMCC 1.3365T. However, the numbers of genes putatively in genome of HX-7-19T such as photosynthesis and metabolisms of potassium, iron, and nitrogen were lower than those of R. blasticus CGMCC 1.3365T.

Table 1 Genome characteristics of strain HX-7-19T and Rhodobacter blasticus CGMCC 1.3365T
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In addition, the ANI and dDDH values between strain HX-7-19T and R. blasticus CGMCC 1.3365T were 76.2% and 20.3%, respectively. Previous study indicated that the threshold values of ANI and dDDH were 95% and 70% generally accepted for bacterial species delineation, respectively [24]. Thus, strain HX-7-19T should represent a novel species according to the results in ANI and dDDH.

Phenotypic and Physiological Characteristics

Individual cell of strain HX-7-19T were rod-shaped with size 0.6–0.8 μm wide and 1.5–3.2 μm long (Fig. S1), and was non-motile and Gram-staining-negative. The colonies were yellowish. The growth of strain HX-7-19T was observed at lower than 1.0% of NaCl (optimum 0%, w/v), temperature at 15–42 °C (optimum at 30 °C), and pH 6.0–8.5 (optimum at 7.0–7.5) (Table 2). The phenotypic and physiological characteristics comparison between strain HX-7-19T and R. blasticus CGMCC 1.3365T were presented at Table 2.

Table 2 Different phenotypic characteristics between strains HX-7-19T and Rhodobacter blasticus CGMCC 1.3365T
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The strain HX-7-19T was able to grow chemo-organoheterophically and photo-organoheterotrophically; whereas it could not grow chemo-lithoautotrophically and fermentative. These nutritional characteristics of strain HX-7-19T was consistent with that of genus Rhodobacter. The photosynthetically grown cell suspension was yellowish brown. The whole-cell absorption spectrum of strain HX-7-19T in sucrose solution (60%, w/v) gave absorption maxima at 376, 485, 683, 755, and 848 nm (Fig. S2). This result indicated that cells of strain HX-7-19T contained bacteriochlorophyll a and pigments [6]. Previous study indicated that the major carotenoids of R. blasticus CGMCC 1.3365T were spheroidene and spheroidenone [5]. HPLC analysis indicated that strains HX-7-19T and the closely related species R. blasticus CGMCC 1.3365T showed the same pattern for major carotenoids (Fig. S3) implying the presence of spheroidene and spheroidenone as major cartenoids [6]. In addition, both strains have the photosynthetic genes pufL and pufM (Fig. S4 and S5), whereas other genus of the family Rhodobacteraceae have not genes pufL and pufM. Their presence confirm the affiliation of strain HX-7-19T to Rhodobacter.

Chemotaxonomic Analysis

As shown in Table 3, the major fatty acids (> 5%) of strain HX-7-19T were C18:1 ω7c (65.98%), C18:1 ω7c 11-methyl (16.69%), and C18:0 (6.25%), while those of R. blasticus CGMCC 1.3365T were C18:1 ω7c (74.92%), C18:0 3–OH (7.21%), and C16:0 (6.55%). On the whole, the fatty acids profile of strain HX-7-19T was similar to that of R. blasticus CGMCC 1.3365T. However, strain HX-7-19T possessed relatively higher amounts of C18:0 and C18:1 ω7c 11-methyl, and lower amounts of C18:0, C18:0 3–OH, and C16:0. The polar lipid profile of strain HX-7-19T is composed of phosphatidylglycerol (PG), diphosphatidyl-glycerol (DPG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), one unknown aminolipid, one unidentified phospholipids, and one unidentified lipids (Fig. S8). The major respiratory quinone of strain HX-7-19T was quinone Q-10, which was coincident with other type strains in the genus Rhodobacter.

Table 3 Cellular fatty acid compositions (percentages) of strain HX-7-19T and related type strain Rhodobacter blasticus CGMCC 1.3365T
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Taxonomic Conclusion

Strain HX-7-19T represents a novel species within the genus Rhodobacter based on the distinct above phenotype and genotype analysis, for which the name Rhodobacter kunshanensis sp. nov. is proposed.

Description of Rhodobacter kunshanensis sp. nov.

Rhodobacter kunshanensis (kun.shan.en’sis. N.L. masc. adj. kunshanensis of or pertaining to Kunshan city, Jiangsu province, China, from where the type strain was isolated).

Cells are non-motile, Gram-staining-negative, and rod-shaped (0.6–0.8 × 1.5–3.2 μm). The colony is yellowish. The growth of strain HX-7-19T was observed at lower than 1.0% of NaCl (optimum 0%, w/v), temperature at 15–42 °C (optimum at 30 °C), and pH 6.0–8.5 (optimum at 7.0–7.5). Growth occurs under anaerobic conditions in the light (photo-organoheterotrophy) or under aerobic conditions in the dark (chemo-organoheterotrophy). The color of phototrophic culture is yellowish brown. Bacteriochlorophyll a, spheroidene and spheroidenone are the photosynthetic pigments. The genomic DNA contains pufL and pufM genes. In the API ZYM system, positive for activities of alkaline phosphatase, esterase (C4), leucine arylamidase, valine arylamidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase. In addition, esterase lipase (C8), lipase (C14), and α-glucosidase are weakly positive. In API 20NE system, 4-nitrophenyl β-d-galactopyranoside and hydrolysis of esculin are positive. d-glucose, d-mannose, capric acid, and trisodium citrate are assimilated. In API 50CH system, acid produced from erythritol, d-ribose, l-xylose, d-glucose, d-fructose, methyl α-d-glucopyranoside, arbutin, inulin, and potassium 5-ketogluconate. In Biolog GENIII tests, positive for α-d-glucose, d-melibiose, l-galactonic acid lactone, d-glucuronic acid, gentiobiose, d-fructose-6-PO4 and tetrazolium violet, negative for d-raffinose, l-arginine, l-lactic acid, l-aspartic acid, and l-glutamic. The polar lipid profile consists of phosphatidylglycerol (PG), diphosphatidyl-glycerol (DPG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), one unknown aminolipid, one unidentified phospholipids, and one unidentified lipids. The major fatty acids are C18:1 ω7c, and C18:1 ω7c 11-methyl. The major respiratory quinone is quinone Q-10. The DNA G + C content is 65.9 mol%.

The type strain HX-7-19T (= KCTC 72471T = CCTCC AB 2020148T) was isolated from activated sludge of an abandoned herbicide manufacturing plant (Kunshan city, Jiangsu province, China).