Introduction

Siderophore are a group of small molecular compounds that chelate ferric iron (Neilands 1995). Microorganisms synthesize and secrete siderophores to acquire iron, because iron is highly insoluble in aerobic environments (Ferguson et al. 1998). Microorganisms which synthesize siderophores with high affinity of iron can be used as biocontrol agency to inhibit plant pathogenic fungi and bacteria by iron competition (Sayye and Chincholkar 2009). Strain HYST was isolated from the water of East Lake (also called Donghu Lake) of Wuhan in China, and was named by its ‘high-yielding of siderophore’ phenotype as determined by a universal siderophore detection assay (Gao et al. 2012; Yu et al. 2014). Strain HYST inhibited growth of the plant pathogenic bacteria Xanthomonas campestris pv. Badrii on LB medium which suggested strain HYST is a biocontrol agency candidate (unpublished).

For the convenience of analysis of siderophores and biocontrol related characteristics, the genomic DNA of strain HYST has been sequenced, and thereby generated a 5,639,475 bp draft genome sequence (Gao et al. 2012). By searching multiple nucleotide and protein databases, 4,556 of 4,955 total predicted protein coding genes found homologs with several strains of genus Pseudomonas, therein 1,856 genes, 1,191 genes and 1,059 genes shared highest similarity with strains of Pseudomonas putida, Pseudomonas fluorescens and Pseudomonas entomophila respectively. The results indicated distinct taxonomic standing of strain HYST despite its close relationship with species of the genus Pseudomonas. Here, we have performed the multilocus phylogenetic analysis based on four house keeping genes together with genomic DNA sequence comparative analysis, phenotypic and chemotaxonomic analyses in order to assess the taxonomic affiliation of strain HYST.

Materials and methods

Bacterial strains

Chrome azurol S (CAS) medium described by Neilands (1995) was used for the isolation of strain HYST from the samples of water of East Lake of Wuhan in China. Strain HYST was grown well on iron-poor modified King’s B medium (Yu et al. 2014) and Luria–Bertani (LB) medium. Reference strains P. putida ATCC 12633T, Pseudomonas vranovensis DSM 16006T, Pseudomonas fuscovaginae DSM 7231T and Pseudomonas asplenii DSM 17133T were obtained from American Type Culture Collection (ATCC) and German Collection of Microorganisms and Cell Cultures (DSMZ) respectively. All strains were grown on LB medium at 28 °C.

Phylogenetic analysis

The 16S rRNA sequence of strain HYST was retrieved from its genome, and then compared with sequences of 16S rRNA gene sequences of the type strains database from EzTaxon server (Chun et al. 2007). Species which have similarity higher than 97.00 % with strain HYST were used for multilocus sequence analysis (MLSA). Three more species, Pseudomonas aeruginosa DSM 50071T, Pseudomonas anguilliseptica NCMB 1949T and Pseudomonas straminea IAM 1598T were also included in the analysis. Cellvibrio japonicus Ueda107 was used as outgroup. The accession numbers of sequences used for MLSA and correspondent references are shown in Table 1.

Table 1 Accession numbers and correspondent references of gene sequences used for phylogenetic analysis in this study
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A series of individual trees of the 16S rRNA, gyrase beta subunit (gyrB), beta subunit of the RNA polymerase (rpoB) partial genes, sigma 70 subunit of RNA polymerase partial genes (rpoD) as well as concatenated gene tree of these four genes were constructed following the methods described in Mulet et al. (2008, 2010). Phylogenetic analysis was performed using the software package MEGA version 6.0 (Tamura et al. 2013) after multiple alignment of the data via CLUSTALW (Larkin et al. 2007). Phylogenetic trees were constructed by neighbour-joining (NJ) (Saitou and Nei 1987) algorithms and evolutionary distances were calculated with Jukes–Cantor method (Jukes and Cantor 1969) and the topology of the NJ tree was evaluated by bootstrap analysis on the basis of 1,000 replications (Felsenstein 1985).

Genome comparison

DNA–DNA hybridization experiments were performed between strain HYST and the type strain of the phylogenetically most closely related P. putida ATCC 12633T, P. vranovensis DSM 16006T, P. fuscovaginae DSM 7231T and P. asplenii DSM 17133T, using the microplate method as described elsewhere (Ezaki et al. 1989; Xie and Yokota 2003).

Average nucleotide identity (ANI) values as the index of whole-genome sequence similarity were calculated as described by Goris et al. (2007). The genomic sequence from one of the genomes in a pair (‘query’) was cut into consecutive 1,020 nt fragments, and then used to search against the whole genomic sequence of the other genome in the pair (‘the reference’) by using the BLASTN algorithm (Altschul et al. 1997). The ANI between the query genome and the reference genome was calculated as the mean identity of all BLASTN matches that showed more than 30 % overall sequence identity over an alignable region of at least 70 % of their length. Therefore, only homologous DNA fragments were considered in the calculations. Reverse searching, i.e., in which the reference genome is used as the query, was also performed to provide reciprocal value.

Morphological, physiological and biochemical characterization

Cell morphology was examined using phase-contrast (Olympus BX51) and transmission electron (FEI Tecnai G2) microscopes (TEM) using cells grown for 12 h. Gram staining of the cells was carried out according to the Gram staining procedure described by Doetsch (1981). Growth at different temperatures (4, 16, 22, 30, 37, 45 and 50 °C) was investigated for 2 days, with growth assessed based on the occurrence of visible colonies on agar. Salt tolerance was tested on modified LB medium in which the NaCl concentration (w/v) was adjusted to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 % respectively. Fluorescent pigment formation was observed on King medium B (King et al. 1954). Hydrolysis of gelatin was investigated according to Stanier et al. (1966). Additional physiological and biochemical characteristics were determined using API 20NE and Biolog GN2 MicroPlate according to the manufacturers’ instructions. All tests were performed using the reference strains in one experiment.

Chemotaxonomic characterization

For respiratory quinones identification, cells were lyophilized and respiratory quinones component were extracted following the method of Collins et al. (1977). Samples were analyzed by HPLC as described by Xie and Yokota (2003).

For analysis of cellular fatty acids, 2 mg late-logarithmic phase cells of strain HYST and the reference strains were collected. The methods used for harvesting, saponification, methylation and extraction of cellular fatty acids followed the protocols of the Sherlock Microbial Identification System (MIDI) version 6.0. Separation and identification of fatty acid methyl esters was performed using a Agilent 6890N gas chromatograph, with MIDI Sherlock TSBA6 (version of the database) (Sasser 1990).

Results and discussion

Phylogenetic analysis

The 1,538 nt length 16S rRNA sequence of strain HYST was used as query sequence to search the type strain 16S rRNA gene sequences database using EzTaxon server. There were thirty-seven type strains of Pseudomonas species sharing similarity higher than 97 % with strain HYST, which represented species of different intrageneric groups except group P. aeruginosa, P. anguilliseptica and P. straminea (Mulet et al. 2010). In order to reconfirm the taxonomic standing of strain HYST within the genus Pseudomonas, the thirty-seven species together with P. aeruginosa DSM 50071T, P. anguilliseptica NCMB 1949T and P. straminea IAM 1598T were employed in MLSA of housekeeping core genes as recommended for new pseudomonas species description (Mulet et al. 2010). Following the approach of Mulet et al., a series of individual phylogenetic trees of four genes (16S rDNA, gyrB, rpoD and rpoB) and four genes concatenated tree have been reconstructed as shown in Fig. 1. The highest gene sequence similarity of four genes concatenated analysis was found to be 95.47 % between strain HYST and P. vranovensis DSM 16006T, and similarities between strain HYST and other reference type strains are lower than 91.70 %. Since the recommended threshold value for species discrimination is 97 % (Goris et al. 2007), strain HYST is suggested to represent a novel species of the genus Pseudomonas.

Fig. 1
figure 1figure 1figure 1

Neighbour-joining phylogenetic trees based on a the 16S rRNA gene; b the gyrB gene; c the rpoD genes; d the rpoB gene; and e four genes concatenated. Distance matrices were calculated by the Jukes–Cantor method. Bootstrap values (1,000 replications) are shown as percentages at each node only if they are 50 % or greater

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Genome comparisons

DNA–DNA hybridization experiments were performed between strain HYST and P. putida ATCC 12633T, P. vranovensis DSM 16006T, P. fuscovaginae DSM 7231T and P. asplenii DSM 17133T. The results are shown in Table 2. The highest DNA–DNA hybridization value was found to be 58 % between strain HYST and P. vranovensis DSM 16006T, which is lower than the threshold 70 % threshold value routinely applied for species discrimination (Wayne et al. 1987).

Table 2 DNA–DNA hybridization relatedness values and gene sequence similarities between strain HYST and closely related Pseudomonas species
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Calculation of ANI of whole-genome sequences is an approach offered by Goris et al. (2007) to accurately replace DNA–DNA hybridization for strains for which genome sequences are available. Recently it is reported that the recommended cut-off value of 70 % DDH for species delineation is corresponded to 95 % ANI (Kim et al. 2014). According to the MLSA and DNA–DNA hybridization data, strain HYST and P. vranovensis DSM 16006T are most closely related. Therefore ANI of whole-genome sequences has been calculated. Since P. putida ATCC 12633T shared the highest similarity with strain HYST based 16S rRNA sequence, the ANI between these two strains is also calculated. The ANI of strain HYST to P. vranovensis DSM 16006T and P. putida ATCC 12633T are 85.25 and 80.96 %, respectively. Both of the ANI values are lower than the 95 %, thus confirming separate taxonomic standing of strain HYST.

Morphological, physiological, and biochemical characteristics

Strain HYST was determined to be Gram-stain negative, non-spore-forming rods (approximately 0.6–1.1 µm wide and 1.7–2.5 µm long) with polar flagella as shown in Fig. 2. Colonies on LB agar are circular and smooth, and the diameter is about 2 mm after 24 h cultivation. Growth was found to occur at 4–37 °C and 0–5 % (w/v) of NaCl. Differential biochemical characteristics of strain HYST are listed in Table 3. Among those characteristics, hydrolysis of gelatin and utilization of sucrose could be typical feature to distinguish HYST from its closest phylogenetic relative species.

Fig. 2
figure 2

Transmission electron microscopy of a negatively stained cell of Pseudomonas donghuensis HYST, showing polar flagella as observed in an FEI Tecnai G2 electron microscope

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Table 3 Characteristics that differentiate strain HYST and closely related Pseudomonas species
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Chemotaxonomic characteristics

The predominant isoprenoid quinone was identified as Q-9, and the minor isoprenoid quinone as Q-8. This isoprenoid quinone composition is typical for the genus Pseudomonas (Collins and Jones 1981). The major cellular fatty acids were identified as C16:0, C17:0 cyclo, Summed feature 3 (C16:1 ω7c or/and C16:1 ω6c) and Summed feature 8 (C18:1 ω7c or C18:1 ω6c) as defined by the MIDI system. The fatty acid compositions of strain HYST and most closely related phylogenetic neighbours are shown in Table 4. Some qualitative and quantitative differences in fatty acid contents are observed between strain HYST and studied species. The proportions of C10:0 and C12:1 3-OH was found to be significantly higher in strain HYST compared to those in references strains.

Table 4 Cellular fatty acid composition of strain HYST and reference strains
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Conclusion

On the basis of the results of phylogenetic analysis based on 16S rRNA gene sequence similarities, MLSA, genomic comparison, phenotypic and chemotaxonomic data presented in this study, it can be concluded that strain HYST represents a new species of the genus Pseudomonas, for which the name Pseudomonas donghuensis sp. nov. is proposed.

Description of Pseudomonas donghuensis sp. nov.

Pseudomonas donghuensis (N. L. fem. adj. dong. hu’ ensis, pertaining to Donghu, where the type strain was isolated).

Cells are Gram-negative, non-spore-forming rods (approximately 0.6–1.1 µm wide and 1.7–2.5 µm long) with polar flagella. Colonies on LB agar are circular and smooth, and the diameter is about 2 mm after 24 h cultivation. Growth occurs at 4–37 °C. NaCl concentrations for growth are 0–5 % (w/v). Fluorescein is produced on King B medium oxidase positive. Gelatin is hydrolyzed. According to API 20NE reduction of nitrate is reduced, d-glucose, potassium gluconate, capric acid, malic acid, trisodium citrate and phenylacetic acid are utilized. Positive for Biolog GN2 MicroPlate substrates: Tween 40, Tween 80, N-Acetyl-d-Glucosamine, l-Arabinose, d-Fructose, α-d-Glucose, Sucrose, Methyl pyruvate, mono-Methyl-Succinate, Acetic acid, Cis-Aconitic acid, Citric acid, Formic acid, d-Gluconic acid, α-Hydroxy, Butyric acid, β-Hydroxy, Butyric acid, p-Hydroxy phenylacetic acid, α-Keto glutaric acid, α-Keto valeric acid, d,l-Lactic acid, Propionic acid, Quinic acid, Succinic acid, Bromo succinic acid, Succinamic acid, l-Alaninamide, d-Alanine, l-Alanine, l-Alanyl-glycine, l-Asparagine, l-Aspartic acid, l-Glutamic acid, Glycyl-l-Glutamic acid, l-Histidine, Hydroxy-l-Proline, l-Leucine, l-Ornithine, l-proline, l-Pyroglutamic acid, d-Serine, l-Serine, l-Threonine, d,l-Carnitine, γ-Amino Butyric acid, Urocanic acid, Inosine, Uridine, Putrescine, 2-Aminoethanol and Glycerol. Major isoprenoid quinone is Q-9, and the minor isoprenoid quinone is Q-8. The major cellular fatty acids are C16:0, C17:0 cyclo, Summed feature 3 (C16:1 ω7c or/and C16:1 ω6c) and Summed feature 8 (C18:1 ω7c or C18:1 ω6c).

The type strain is HYST ( = CCTCC AB 2012141T = NRRL B-59108T).