Introduction

The genus Alsobacter of the family Alsobacteraceae, class Alphaproteobacteria was first proposed by Bao et al. (2014). At the time of writing, the genus Alsobacter comprises two species with validly published names. Species of this genus have been reported from a garden soil sample (Bao et al. 2014) and a paddy soil sample (Sun et al. 2018). According to the former studies, Alsobacter species are Gram-staining negative, aerobic and rod-shape, containing Q-10 as the respiratory ubiquinone (Bao et al. 2014; Sun et al. 2018). In this study, we describe a new isolate which represents a novel species of this genus based on the analysis of morphological, phenotypic, genotypic, and phylogenetic characteristics.

Estuaries are the interaction zones between oceans and terrestrials, whose eco-environment health are essential to the sustainable development of human habitation and social economy. In order to investigate the cultivable bacteria community in the Pearl River estuary, surface sediment samples were collected from Pearl River estuary, Guangzhou, Guangdong, China. During this study, one novel isolate SYSU M60028T was cultivated from a sediment sample.

Material and method

Sample collection and isolation

The surface sediment samples (10 cm) were collected at the depth of 3 m from the Pearl River, Guangdong, China, and transferred by a sterile homogeneous bag. The temperature and pH of the samples were 28 °C and pH 7.5, respectively. The sediment samples were diluted 103- fold with sterile distilled water and 0.2 mL final suspension was plated on Reasoner's 2A (R2A; BD) agar (Yeast Extract 0.5 g, Proteose Peptone 0.5 g, Glucose 0.5 g, Soluble Starch 0.5 g, Casamino Acids 0.5 g, Sodium Pyruvate 0.3 g, K2HPO4 0.3 g, MgSO4-7H2O 0.05 g, Distilled Water 1000 ml, Agar 18 g, pH 7.0). The isolation plates were incubated at 28℃ for 7 days. Single colonies were selected and purified on the same medium. The purified strains were preserved as glycerol suspensions (20%, v/v) at − 80 °C for further use. Among the purified isolates, one off-white strain SYSU M60028T showed low 16S rRNA sequences similarities to Alsobacter metallidurans (96.87%) and Alsobacter soli (96.87%), and was selected for further novel species characterization. The basal growth conditions of the strain SYSU M60028T for all experiments were maintained at pH 7.0 and 28℃, unless otherwise stated.

Phenotypic characterization

Cultural characteristics were performed on Luria–Bertani (LB) agar, R2A agar, tryptic soy agar (TSA; Difco), and marine agar 2216E (MA; BD Difco) at 28 °C for 5 days. The microscopic morphological characteristics of the strain SYSU M60028T were observed by transmission electron microscope (JEM–1400FLASH, JEOL, JAPAN). The Gram-reaction was performed by the standard Gram reaction and was confirmed by the non-staining method using 3% KOH (Buck 1982). Growth temperature (4, 10, 15, 20, 25, 28, 30, 37, 45, 55, 60 and 65 °C), pH range (pH 4.0–10.0, at intervals of 1 unit, adjusted by using the buffer system) (Xu et al. 2005), and NaCl tolerance (0, 1, 2, 3, 4, 6, 9, 12 and 15%, w/v) were tested on R2A agar. Oxidase and catalase activities were determined by assessing the oxidation of 1% (w/v) tetramethyl-p-phenylenediamine (Kovacs 1956) and the formation of bubble on addition of 3% H2O2, respectively. Tweens (20, 40, 60, and 80) degradation, H2S production, milk coagulation and peptonization, cellulose, starch, gelatin hydrolysis, anaerobic growth, and urease activity were tested according to the methods previously described (Gonzalez et al. 1978; Smibert Krieg 1994). Other physiological and biochemical characteristics of strain SYSU M60028T were identified by GEN III Microplates system (Biolog, USA), API 20NE and API ZYM kits (bioMérieux) according to the manufacturer’s instructions.

Chemotaxonomy characterisation

Biomass for chemical and molecular studies of strain SYSU M60028T was obtained on R2A agar during the logarithmic growth period (3 days). For polar lipids, strain SYSU M60028T was extracted and identified by two-dimensional TLC on silica gel G60 plates (Merck) as previously described (Collins Jones 1980; Minnikin 1979). The respiratory ubiquinone was extracted from lyophilized cells as described by Collins et al. (1977), purified, and analysed by HPLC as described by Tamaoka et al. (1983). The cellular fatty acid methyl esters were extracted and analysed according to the standard protocol of the Microbial Identification System (Sherlock version 6.1; MIDI database: TSBA6) (Athalye et al. 1985).

Phylogenetic analysis

Genomic DNA extraction and PCR amplification of 16S rRNA gene were carried out according to the method described by Li et al. (2007). The obtained sequences were compared with the corresponding validly published species in the EzBioCloud database (Yoon et al. 2017). The 16S rRNA gene sequences of close related type strains were aligned using CLUSTAL X software package (Thompson et al. 1997) and were used for constructing the phylogenetic trees by MEGA X software (Kumar et al. 2018). Algorithms based on neighbour-joining (Saitou Nei 1987), maximum-likelihood (Felsenstein 1981), and maximum-parsimony (Fitch 1971) methods were used for generation of the phylogenetic trees. Kimura’s two-parameter model was used to calculate evolutionary distance matrices of the phylogenetic trees (Kimura 1980). Stability of the phylogenetic trees was evaluated by bootstrap analysis with 1000 replications (Felsenstein 1985). Chelatococcus reniformis B2974T was used as an outgroup.

Genome sequencing, annotation, and analysis

The whole genome sequencing of strain SYSU M60028T was performed by using Illumina NovaSeq platform at the Guangdong Microbial Culture Collection Center (GDMCC, Guangdong, China). Reads of each data sets were filtered, and high quality paired-end reads were assembled using readfq (version 10), SOAP denovo (version 2.04), and SPAdes (Bankevich et al. 2012; Li et al. 2008, 2010). The quality of the genomes was examined using CheckM v.1.0.7 (Parks et al. 2015). The COG database (Galperin et al. 2014) and KEGG database (Kanehisa Sato 2020; Kanehisa et al. 2022) were used to annotate the genome sequences. The secondary metabolite biosynthetic gene clusters were annotated by antiSMASH (Blin et al. 2021). The Average nucleotide identity (ANI) was calculated using JSpecies (http://jspecies.ribohost.com/jspeciesws/) (Richter et al. 2016). The digital DNA-DNA hybridization analysis was performed by using the DSMZ Genome-to-Genome Distance Calculator platform (http://ggdc.dsmz.de/distcalc2.php) (Goris et al. 2007). The phylogenomic tree was constructed according to the method described by Jiao et al. (2021). The multiple sequence alignments (MSAs) were generated by GTDB-Tk software (Chaumeil et al. 2019). The Maximum-likelihood phylogenetic tree was calculated by IQ-Tree with parameters (alrt 1000 bb 1000 nt AUTO) (Nguyen et al. 2015). The best-fit model (LG + F + R4) determined by ModelFinder was well supported by Akaike information criterion (AIC), corrected AIC (AICc), and Bayesian information criterion (Kalyaanamoorthy et al. 2017). The phylogenomic tree was visualized using the online Interactive Tree of Life program version 4.2 finally (Letunic Bork 2016).

Result and discussion

Phenotypic characteristics

Cells of strain SYSU M60028T were Gram-staining negative, aerobic, and had the ability to move by flagella (Fig. S4). Colonies were circular, punctiform and off-white in colour. Cells were short rods with size measuring 0.6–0.7 × 0.9–1.0 μm (Fig. S4). The isolate could be able to grow at pH 6.0–8.0 (optimum, pH 7.0), 25–37 °C (optimum, 28 °C) and in the presence of 0–2% (w/v) NaCl (optimum, 0% NaCl). Strain SYSU M60028T was positive for oxidase activity, α-glucosidase, H2S production, nitrate reduction and trypsin, but negative for milk coagulation and peptonization, Tweens (20, 40, 60, and 80), or catalase activity. The characteristics distinguishing strain SYSU M60028T from closely related strains are listed in Table 1. The detailed physiological characteristics of strain SYSU M60028T are given in the species descriptions below.

Table 1 Differential phenotypic and chemotaxonomical characteristics between strain SYSU M60028T and its closely related type strains in the genus Alsobacter
Full size table

Chemotaxonomical characteristics

The respiratory quinone of strain SYSU M60028T was identified as ubiquinone Q-10. The major cellular fatty acids (> 5%) detected of strain SYSU M60028T were summed feature 8 (C18:1ω7c/ C18:1ω6c, 55.3%), C16:0 (9.0%), and C18:1 ω7c 11-methy1 (5.8%). The cellular polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, one unidentified aminolipid and three unidentified lipids (Fig. S5). The detailed fatty acid profile of strain SYSU M60028T was provided in Table S4.

Phylogenic and phylogenomic analysis

The 16S rRNA gene sequences of strain SYSU M60028T was submitted to GenBank with accession number ON881906. On pairwise comparison with the 16S rRNA gene sequences available in the EzBioCloud database, strain SYSU M60028T showed highest 16S rRNA gene sequences similarities to Alsobacter metallidurans SK200a-9T (96.87%) and Alsobacter soli SH9T (96.87%). The neighbour-joining tree based on 16S rRNA gene sequences showed that strain SYSU M60028T formed a distant clade with Alsobacter metallidurans SK200a-9T and Alsobacter soli SH9T (Fig. 1). An apparently stable lineage was also supported by maximum-parsimony and maximum-likelihood phylogenetic trees (Fig. S1, Fig. S2). Phylogenomic tree (Fig. S3) based on the concatenated alignment of 120 marker genes provided further evidence of this topological structure. Based on the 16S rRNA gene sequences identity and the phylogenetic relationship, Alsobacter metallidurans SK200a-9T and Alsobacter soli SH9T were used as reference type strains.

Fig. 1
figure 1

Neighbour–joining phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strain SYSU M60028T and their closest relatives. Bootstrap values (> 50%) based on 1000 replications are given at the nodes. Bar represents 0.01 substitutions per nucleotide position. Chelatococcus reniformis B2974T (KJ469373) was used as an outgroup

Full size image

Genome analysis

The genome properties of strain SYSU M60028T included 56 contigs amounting to 4,727,519 bp. The G + C content based on DNA genomic was 69.9% (Table S1). The completeness and contamination of the genomic were 98.59% and 0.26%, respectively. The dDDH value comparing strain SYSU M60028T with Alsobacter soli SH9T and Alsobacter metallidurans SK200a-9T were 21.2% and 20.8% (Table S2), which were below the cutoff (70%) recommended as the criterion for interspecies identity (Wayne 1988). The ANI values comparing SYSU M60031T with Alsobacter soli SH9T and Alsobacter metallidurans SK200a-9T were 75.39% and 75.37% (Table S2), further suggesting the strain SYSU M60028T represents a novel species belonging to the genus Alsobacter.

According to the genome annotation results, the genome of the strain SYSU M60028T contained 4,264 protein-coding genes. The number of genes assigned to COG database and KEGG database were 3,304 and 3,732, respectively. COGs (Clusters of Orthologous Groups) categories distributions for the genes were presented in the Table S3. Amino acid transport and metabolism (COG category E) was the most abundant represented CDs (sequence coding for amino acids in protein) of strain SYSU M60028T, followed by general function prediction only (COG category R), function unknown (COG category S), and carbohydrate transport and metabolism (COG category G). According to the antiSMASH database, T3PKS, NRPS, NRPS-like and terpene gene clusters were four main clusters of the strain SYSU M60028T, which were responsible for the biosynthesis of secondary metabolites (Table S5). Strain SYSU M60028T had the potential to produce terpene, which offered new insight into biosynthesis of terpene in bacteria.

According to the annotation results of KEGG analysis, strain SYSU M60028T contained 14 genes which were relevant to the nitrogen cycle, including dissimilatory nitrate reduction, denitrification, and nitrification. Genome annotation results suggested that strain SYSU M60028T contained 4 genes (narGHI, nirK, norBC, and nosZ), which formed a complete denitrification pathway. Compared with type strains, Alsobacter soli SH9T also had the ability of denitrification, while Alsobacter metallidurans SK200a-9T did not have this complete pathway. This result was identified by the positive result of nitrate reduction test in API 20NE kit. Ntr C (a DNA-binding transcriptional regulator) was predicted in strain SYSU M60028T. The previous studies had indicated that Ntr C participated in nitrogenous compounds utilization, biological nitrogen fixation, the biosynthesis of biopolymer and maintenance on carbon–nitrogen balance (Hervas et al. 2008). Therefore, the strain might involve in nitrogen-transforming reactions and remove nitrogen, which meant this strain could play an important role in nitrogen cycle and reducing the eutrophication of nearshore waters (Falkowski et al. 2008). Strain SYSU M60028T also contained a complete assimilatory sulfur pathway (cysND, cysC, cysH, and cysJI), while Alsobacter metallidurans SK200a-9T and Alsobacter soli SH9T did not have this complete pathway. This result was consistent with the positive result of H2S production test and suggested the strain might reduce sulfate to sulfide, use sulfate as a terminal electron acceptor for the degradation of organic compounds, resulting in the production of sulphide, and kept the dynamics of sulfur cycle in balance (Muyzer et al. 2008). In addition, the genome contained 25 genes related to flagellar assembly. The diffusion of cells in semi-solid medium, and the flagellar observed by transmission electron microscope (Table S3) further supported the result of genome analysis.

Conclusion

The results of the phylogenetic analysis showed the strain SYSU M60028T was a member of Alsobacter. Combining the morphological and chemotaxonomic characteristics, the strain SYSU M60028T could be distinguished from other Alsobacter species. For example, the proportions of fatty acids and polar lipid composition or the utilization of carbon and nitrogen source of strain SYSU M60028T are significantly different from those of the related species. Compared with Alsobacter metallidurans SK200a-9T (4630) and Alsobacter soli SH9T (4751), Strain SYSU M60028T contained the least number of protein-coding genes (4290). According to the annotation results of KEGG analysis, strain SYSU M60028T contained the complete assimilatory sulfur pathway (cysND, cysC, cysH, and cysJI), while Alsobacter metallidurans SK200a-9T and Alsobacter soli SH9T did not have this complete pathway. Strain SYSU M60028T contained 4 genes (narGHI, nirK, norBC, and nosZ), which were formed a complete denitrification pathway. Compared with type strains, Alsobacter soli SH9T also had the ability of denitrification, while Alsobacter metallidurans SK200a-9T did not have this complete pathway. Thus, based on above results, we propose the strain SYSU M60028T as a novel species of the genus Alsobacter, for which the name Alsobacter ponti sp. nov. is proposed.

Description of Alsobacter ponti sp. nov

Alsobacter ponti (pon’ti. L. gen. masc. n. ponti, of the sea).

Cells are Gram-staining negative, aerobic, motile, and short rods (0.6–0.7 × 0.9–1.0 μm). Colonies on R2A agar are off-white, circular, and smooth. Able to grow at pH 6.0–8.0 (optimum, pH 7.0), 25–37 °C (optimum, 28 °C) and in the presence of 0–2% (w/v) NaCl (optimum, 0% NaCl). Positive for oxidase activity, but negative for milk coagulation and peptonization, Tweens (20, 40, 60, and 80), or catalase activity. Positive for sterase (C4), esterase lipase (C8), α-chymotrypsin, trypsin, alkaline phosphatase, cystine arylamidase, valine arylamidase and leucine arylamidase. Negative for hydrolysis of urea, cellulose and gelatin, reduction of nitrates to nitrites, lipase (C14), acid phosphatase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, and α-mannosidase. The predominant respiratory quinone is Q-10. The cellular polar lipids are phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, one unidentified aminolipid and three unidentified lipids. The major fatty acids (> 5% of total) are summed feature 8, C16:0 and C18:1 ω7c 11-methy1. The G + C content of the genome DNA is 69.9%.

The type strain, SYSU M60028T (= CGMCC 1.19341T = KCTC 92046T) was isolated from a sediment sample collected from the Pearl River estuary, Guangdong, China. The 16S rRNA gene sequences of strain SYSU M60028T has been submitted to GenBank with accession number ON881906. The accession numbers of raw data and assembly genome are SRR23021099 and JANCLU000000000, respectively.