Introduction

The genus Myceligenerans was first described from an alkaline salt marsh soil in western China (Cui et al. 2004). It belongs to the family Promicromonosporaceae under the suborder Micrococcineae. Presently, there are four reported species with validly published names (https://lpsn.dsmz.de/genus/myceligenerans). There is no report of the presence of Myceligenerans from mangrove intertidal sediment.

The objective of the present study was to identify and characterize a new species of Myceligenerans isolated from intertidal sediment of Indian Sundarbans mangroves.

Materials and methods

Study area

Sundarbans cover an area of approximately 10,000 km2 (Gopal and Chauhan 2006), located between India and Bangladesh at 21°40′N and 22°09′N latitude, and 88°01′E and 89°06′E longitude. It is formed on the delta of Ganga–Brahmaputra–Meghna riverine systems and faces the Bay of Bengal. Globally, this is the largest contiguous mangrove ecosystem traversed by innumerable estuaries and creeks. The eastern part of Indian Sundarbans has been declared as the Sundarbans Biosphere Reserve (SBR) for conservation of rich biodiversity. The SBR is divided into core zone, buffer zone, and transition zone. The collection of sediment as part of this study was undertaken from a predesignated sampling station Stn_SBR_N3 (21°56′40.74"N, 88°40′34.98"E), located at the confluence of River Matla and River Thakuran within the buffer zone of SBR. The collection of intertidal sediment was undertaken in February, 2015 in low tide using a hand-held corer (length 10 cm, diameter 2.5 cm). At the time of collection, in situ salinity was recorded by a hand-held refractometer (Erma Inc., Japan). The unfixed sediment was immediately transferred to the laboratory for downstream analyses.

Isolation of actinobacteria

Isolation of Actinobacteria was undertaken from 0 to 2 cm vertical profile of collected sediment core. Briefly, 0.5 gm sediment representing the vertical profile was suspended in 10 mL of 0.22 μm filtered and double autoclaved seawater (salinity-20; based on in situ salinity measurement). After vigorous mixing, the suspension was allowed to settle for 1 h under room temperature (RT) (25 ± 1 °C). From the supernatant, 2, 5 and 10 μL were pipetted and spread plated on petridishes (90 mm) (Tarsons, India) containing actinomycetes isolation agar (AIA) medium (HiMedia, India) supplemented with 0.5% autoclaved glycerol (Merck, India). The petridishes were subsequently incubated at room temperature (RT) (25 ± 1 °C) for 15 days. Pure strains were selected based on morphological characteristics, Gram staining, and biochemical characterizations. Following this, an isolate (referred as I2) was selected based on unusual morphological features for subsequent polyphasic characterizations.

Morphological characterization of I2 isolate

Cell and colony morphology were observed following Gram staining using bright field microcopy (BX53, Olympus Corporation, Japan) attached to a CCD camera with the help of CellSens software (Olympus). Besides, differential interference contrast (DIC) microscopy was also undertaken. Field emission scanning electron microscopy (FESEM) was undertaken in Zeiss SUPRA55VP (Carl Zeiss AG, Germany) to confirm morphological characteristics based on published protocol (Singh and Bhadury 2019).

Growth experiments of I2

Culture conditions, namely, pH (range: 4, 5, 6, 7, 8, 9 and 10), temperature (4, 10, 15, 20, 25, 30, 35, 40, 45 and 50 °C) and salinity (5, 10, 15, 20, 25, 30, 35, 40 and 45 ppt) were examined to determine optimum growth conditions of I2.

Biochemical characterization of I2

Biochemical tests of the isolate were performed at salinity 20, pH 7, and room temperature (RT).

Growth of I2 on different substrates

The ability of I2 to utilize different carbon sources was determined using HiCarbo™ Kit (HiMedia, India) as per manufacturer’s instructions. Culture characteristics was also determined after incubation at RT for 7 days in ISP1-7 (International Streptomyces Project; [Shirling and Gottlieb 1966]) (HiMedia, India), actinomycetes isolation agar, Zobell marine agar (HiMedia, India) Czapek’s agar, nutrient agar, and LB agar. The colors of substrate and aerial mycelia were determined by comparison with the ISCC-NBS color charts (Kelly 1964).

Antibiotic susceptibility test of I2

Antibiotic susceptibility of I2 was observed by well diffusion method against ampicillin, streptomycin and chloramphenicol (final concentration each 10 μg).

Chemotaxonomic analysis

I2 was grown on Trypticase soy agar (salinity 20, pH 7) at 37 °C for 7 days. Cellular fatty acid composition was determined (Sasser 1990) using the Microbial Identification System (MIDI Sherlock). Whole-cell sugars, structure of peptidoglycan, menaquinones, and polar lipids were determined based on published protocols (Schleifer and Kandler 1972; Staneck and Roberts 1974; Minnikin et al. 1984; Nguyen and Kim 2017).

Genomic DNA extraction

Cells were harvested in log phase and genomic DNA (gDNA) was extracted following published protocol (Bostrӧm et al. 2004).

PCR amplification and DNA sequencing

Near complete 16S rRNA gene sequence was amplified from gDNA of I2 using published primers (Lane 1991). PCR conditions were followed as per published protocols (Ghosh and Bhadury 2019). Ketosynthase domain of polyketide synthases (PKSs) was amplified using published primers (Moffitt and Neilan 2001). Beta subunit of ATP synthase and RNA polymerase genes were amplified following published protocols (Gaunt et al. 2001; Dahllöf et al. 2000). Purified PCR products were cloned and sequenced using Sanger approach. Chromatograms were checked using BioEdit v7.0 (Hall 1999) for any ambiguity or error before undertaking downstream analyses.

Molecular phylogenetic analyses

The 16S rRNA sequence of I2 was aligned to those of representatives of Promicromonosporaceae within the phylum Actinobacteria. The TrN + I + G were selected as the best model for 16S rRNA phylogeny using jModelTest V2.1.2 (Darriba et al. 2012). For phylogeny, maximum likelihood (ML) method was used in MEGA X (Kumar et al. 2018). To infer phylogeny of ketosynthase domain of PKS (amino acid level), ML incorporating Jones–Taylor–Thronton (JTT) model was used. A concatenated tree of atpD and rpoB (amino acid level) was constructed using ML incorporating JTT model. The generated sequences have been submitted to GenBank and their accession numbers are MH359322 (16S rRNA), MH359323 (atpD), MH359324 (PKSs) and MH359325 (rpoB).

DNA G + C content determination

The G + C content in gDNA was determined following spectrophotometric method (Marmur and Doty 1962) in UV–Visible Spectrophotometer (U-2900, Hitachi, Japan).

DNA–DNA hybridization

DNA–DNA relatedness values (ΔTm) of I2 and Myceligenerans xiligouense XLG9A10.2 were determined spectrophotometrically based on published protocol (De Ley et al. 1970).

Whole-genome sequencing

Genome sequencing library was prepared using the Illumina-compatible SureSelectQXT whole-genome preparation kit (Agilent, USA). Fragmented and adapter-tagged gDNA was amplified and sequenced on Illumina MiSeq platform. The sequence data were checked using FastQC and adapters were trimmed using Cutadapt (Martin 2011). The pair-end reads were assembled using Unicycler (Wick et al. 2017).

Whole-genome sequence annotation

Genomic annotation was carried out using Prokka (Seemann 2014). Genomic islands were predicted using IslandViewer 4 (Bertelli et al. 2017). Genomic relatedness with type strains was determined using OrthoANIu algorithm (http://www.ezbiocloud.net/tools/ani) (Yoon et al. 2017). Digital DDH values were calculated by applying Formula 2 (identities/HSP length) in Type Genome Server (Meier-Kolthoff and Göker 2019). Average amino acid index (AAI) between I2 with type strains was determined using AAI-profiler (http://ekhidna2.biocenter.helsinki.fi/AAI/) (Medlar et al. 2018). In silico phenotyping was undertaken using Traitar (Weimann et al. 2016). The genome of I2 was aligned into circular map using CGView server (Grant and Stothard 2008). The genome sequence has been deposited to GenBank/ENA/DDBJ under the accession number JABBYC000000000.

Results and discussion

At the time of sediment collection, in situ salinity was recorded as 20 ppt. Fifteen pure bacterial strains were isolated and subjected to morphological and biochemical characterizations. The isolate I2 was selected based on unique morphology.

Morphological characterization of I2

I2 was Gram positive. Mycelia along with coccoid- and rod-shaped elements were observed using bright field and DIC approaches (Figs. S1a and b) as well as confirmed by FESEM (Fig.S2). Comparison of morphology using FESEM revealed similarities between isolate I2 and Myceligenerans xiligouense XLG9A10.2.

Growth experiments of I2

I2 exhibited growth between 10–40 °C temperature (optimum—25 °C), salinity 10–40 (optimum—20) and pH 5–9, (optimum—7). The doubling time of this isolate was calculated as 11.5 h.

Biochemical characterization of I2

I2 was found to be non-motile actinobacterium, exhibited positive and negative results for biochemical tests as detailed in Table 1. The isolate was able to hydrolyze both starch and gelatin.

Table 1 Comparison of biochemical tests and antibiotic susceptibility of I2 with other published species of Myceligenerans. I2 shows characteristic biochemical tests similar to other Myceligenerans spp.
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Growth on different substrates

I2 could utilize different carbon sources at RT (Table 2). The number of filaments decreased (highest in dextrose, lowest in raffinose) with the increase in complexity of carbon source, while the number of rod- and cocci-shaped elements increased (highest in raffinose, lowest in dextrose) (Fig. S3).

Table 2 Comparison of carbon utilization sources by I2 with other published species of Myceligenerans
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The growth of I2 was observed on ISP1-7 (International Streptomyces Project; [8]), actinomycetes isolation agar [9], Czapek’s agar, marine agar, nutrient agar, and LB agar. Aerial mycelia were only observed on ISP4 at RT, salinity 20 and pH 7. The substrate mycelia were well developed, but fragmented into rod-shaped elements. Substrate mycelia was yellowish-white on yeast extract-malt extract (ISP2), glycerol-aspargine agar (ISP5), Tyrosine agar (ISP7), Czapek’s agar and Marine agar; pale yellow on Luria–Bertani agar and Nutrient agar and white on oatmeal agar (ISP3), inorganic salts—starch agar (ISP4) and peptone-yeast extract iron agar (ISP6) media.

Antibiotic susceptibility test of I2

I2 was susceptible to ampicillin and chloramphenicol but resistant to streptomycin. Antibiotic susceptibility results of isolate I2 was compared with other known Myceligenerans spp. (Table 1).

Chemotaxonomic analysis

The predominant fatty acid encountered in I2 were anteiso-C15 (55.81%) and anteiso-C17 (15.45%) (Table 3). Iso-C15 comprised only 6.54% of the total fatty acids which was lower compared to other Myceligenerans spp. (range 18.5–31.4%) [1–5]. Other major fatty acid included iso-C16 (6.50%). Minor fatty acids were represented by iso-C14 (1.49%), iso-C17 (2.42%), C18 (2.35%), iso-C19 (1.07%), anteiso-C19 (1.79%), and C20 (1.57%) (Table 3). Major cell-wall sugars were glucose, galactose, and mannose. Rhamnose was also present. The peptidoglycan of I2 belonged to type A4α, l-Lys–l-Thr–d-Glu. The predominant menaquinone was MK-9 (H4). The polar lipids were represented by diphosphatidylglycerol, one unknown phospholipid and one unknown glycolipid.

Table 3 Comparison of fatty acid methyl ester (FAME) profile of I2 with other published species of Myceligenerans
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Molecular phylogeny

Based on blastn result, 16S rRNA sequence of I2 showed 99% identity (100% query coverage) with published 16S rRNA sequence of Myceligenerans salitolerans strain XHU 5031 (Accession number: NR_132702). The result was also confirmed by RDP-II and SILVA, which showed closest match with the genus Myceligenerans. Based on 16S rRNA phylogeny, isolate I2 clustered with Myceligenerans salitolerans strain XHU 5031, Myceligenerans xiligouense strain XLG9A10.2, Myceligenerans halotolerans strain XJEEM 11063 and Myceligenerans cantabricum strain M-201 (Fig. 1). The sequenced ketosynthase domain (146 amino acids) in I2 showed significant identity with polyketide synthases sequence of Gloeothece sp. strain PCC 6909 (Accession number: AAX44107) (72% identity, 100% query coverage). In PKSs phylogeny, I2 represented part of a clade comprising of Micromonospora sp. CMS I232 (Accession number: AGI61660) and Salinispora arenicola (Accession number: ADZ48591) (Fig. 2). The genes coding for PKSs is thought to be widespread in Actinobacteria; however, only limited number of studies is available within the family Promicromonosporaceae [examples—Promicromonospora aerolata (Yang et al. 2018); Cellulosimicrobium cellulans (Schumann and Stackebrandt 2012)] but not previously reported in the genus Myceligenerans. The multigene concatenated phylogenetic tree of atpD and rpoB placed I2 in a separate subclade compared to known members under Promicromonosporaceae (Fig. 3, Table S1).

Fig. 1
figure 1

Maximum likelihood tree based on 16S rRNA sequences showing relationship of I2 with closely related taxa. The 16S rRNA sequence of Arthrobacter globiformis is used as the outgroup. The scale bar indicates 0.01 substitutions per nucleotide site

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Fig. 2
figure 2

Maximum likelihood tree based on KS domain (PKSs) amino acid sequences showing relationship of I2 with closely related taxa. The KS domain amino acid sequence of Arthrobacter globiformis is used as the outgroup. The scale bar indicates 0.5 substitutions per amino acid site

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Fig. 3
figure 3

Concatenated multigene phylogenetic tree based on amino acid sequences of atpD and rpoB showing relationship of I2 with closely related taxa. The scale bar 0.10 indicates substitutions per amino acid site

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DNA G + C content

DNA G + C content of isolate I2 was 72 mol%, which is comparable with DNA G + C content of other known Myceligenerans spp. (range 71.8–72.4 mol%) (Cui et al. 2004). The value falls within range reported in members representing the family Promicromonosporaceae (70–76 mol%).

DNA–DNA hybridization

The ΔTm between I2 genomic DNA and I2/Myceligenerans xiligouense XLG9A10.2 hybrid DNA was 5 °C and the DNA–DNA relatedness value was 65% (Gonzalez et al. 2005).

Genome analysis

The draft genome of I2 consisted of ~ 5 Mbp which assembled into 145 contigs. The GC content was 71.39%. The genome map of I2 is shown in Fig. 4. Genome analysis indicated the presence of 4366 CDS. Genome sequence indicated the presence of 68 tRNA, 5 rRNA, and 1 tmRNA. The draft genome contained the full length of the 16S rRNA. The GGDC (%), orthoANIu(%), and AAI values of I2 were found to be 28.9%, 77.44% and 0.859, respectively, when compared to genome of Myceligenerans xiligouense strain DSM 15,700 (Table S1). The GGDC, orthoANIu and AAI values indicating relatedness of I2 with other members are also detailed in Table S2. The alignment between genome sequences of I2 with M. xiligouense strain DSM 15700 is shown in Fig. S4. Genome annotation has revealed the presence of a large number of genes linked to amino acids and derivatives (270), carbohydrate metabolism (245), protein metabolism (166), nucleosides and nucleotides (94), metabolism of aromatic compounds (33), cell wall and capsule (24), nitrogen metabolism (8), among others. The genes linked to PKSs pathway were found in the genome of I2 including the presence of polyketide biosynthesis cytochrome P450 (PksS), polyketide biosynthesis protein E (PksE) and phthiocerol/phenolphthiocerol synthesis polyketide synthase type I (PpsA). Besides, a number of genes (14) linked to biotin synthesis, genes such as soj which control sporulation and genes linked to resistance of antibiotics and toxic compounds were also encountered. In silico phenotyping indicated the bacterium to be aerobic, Gram-positive, and spore forming. A summary of the positive and negative in silico phenotyping results is shown in Fig. S5. The position of the genomic islands which might be linked to antibiotic resistance or virulence is shown in Fig. S6. The genome sequence version described in this paper is JABBYC000000000.

Fig. 4
figure 4

Circular genome of the isolate I2

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Taxonomic conclusion

On the basis of morphological, biochemical, phylogenetic, chemotaxonomic and genome analyses, the isolate I2 belongs to the genus Myceligenerans and also represents a new species, Myceligenerans indicum sp. nov. The proposed new species is the first report from a mangrove ecosystem.

Description of Myceligenerans indicum sp. nov.

Myceligenerans indicum (in'di.cum. L. neut. adj. indicum, of India, Indian).

This species is Gram positive, aerobic, and non-motile actinobacterium. The bacterium has well developed substrate mycelia that fragment into cocci- and rod-shaped elements, while aerial mycelia only formed on ISP4 media. Growth occurs at 10–40 °C temperature with optimum temperature at 25 °C, pH 5–9, with optimum at pH 7 and salinity 10–40 with optimum at salinity 20. The bacterium is susceptible to ampicillin (10 μg) and chloramphenicol (10 μg), while resistant to streptomycin (10 μg). Starch, Esculin and gelatin hydrolysis are positive, while nitrate reduction is negative. The isolate was able to utilize xylose, maltose, fructose, dextrose, galactose, raffinose, trehalose, melibiose, sucrose, l-arabinose, mannitol, arabitol, rhamnose, cellobiose, mannose, ONPG and d-arabinose; but was unable to utilize lactose, inulin, sodium gluconate, glycerol, salicin, dulcitol, inositol, sorbitol, adonitol, erythritol, α-methyl-d-glucoside, sorbose, xylitol, malonate and citrate. Major fatty acids present are anteiso-C15, iso-C15, iso-C16, anteiso-C17. Major cell-wall sugars were glucose, galactose and mannose. The peptidoglycan of I2 belonged to type A4α, l-Lys–l-Thr–d-Glu. The predominant menaquinone was MK-9 (H4). The polar lipids present were diphosphatidylglycerol, one unknown phospholipid and one unknown glycolipid. The genomic DNA G + C content was found to be 72 mol%. This new species has the potential ability to produce secondary metabolites as evident from the existence of KS domain of PKS. The genome size is ~ 5 Mbp. Genome annotations have indicated the presence of essential genes such as for utilization of sugars and amino acids, vitamin biosynthesis and capsule formation. In silico analysis showed the organism to use carbon sources including sucrose, l-arabinose, rhamnose, d-xylose, and trehalose for growth. The type strain isolated from the mangrove sediment of Indian Sundarbans is maintained as a pure culture in Microbial Type Culture Collection (MTCC) (MTCC 1291)0 and in Marine Microbial Culture Laboratory, IISER Kolkata, Mohanpur, Nadia, India (PBKA002).