Phylogenetic insight of Nonribosomal peptide synthetases (NRPS) Adenylate domain in Antibacterial potential Streptomyces BDUSMP 02 isolated from Pitchavaram Mangrove

Identification of gene clusters in Streptomyces holds promise for the discovery of regulatory pathways linked to bioactive metabolites. We isolated a broad-spectrum antibacterial potential Streptomyces sp BDUSMP 02 from mangrove sediment. We further found a distinct of phylogeny pattern for NRPS A-domain in the Streptomyces sp BDUSMP 02. The result suggests that Streptomyces sp BDUSMP 02 has the potential to produce a new type of antibacterial compounds belonging to NRPS type.


Background:
In the last five decades, natural compounds produced by actinobacteria have been enormously utilized to develop most of the common antibiotics commercialized by pharmaceutical industries [1]. Given this, isolation of actinomycetes from unexplored marine environment has been attracted particular attention due to their structural diversity and distinct bioactivities of secondary metabolites produced by them [2]. In evidence, Salinispora comes under the genus Actinomycete was first isolated from ocean sediments [3]. Mangrove forests are located in the tidal zones in tropical and subtropical regions [4]. Bissett et al. (2007) reported that mangrove sediments are known to contain high organic content, which favour the rapid development of species diversity corresponding to environmental variation [5]. The exploitation of mangrove actinomycetes for bioactive compounds has been increased dramatically [6][7][8]. Streptomyces sp isolated from mangrove ecosystem have been able to grow in freshwater, brackish water and seawater which suggest that they are adapted to various environmental conditions due to the water current [7]. Besides, it could be a starting point to study the evolution of gene clusters responsible for the biosynthesis of novel antibiotic because of their adaptation to extraordinarily salty and marshy condition [7].
It is evident that gene clusters in Streptomyces likely to encode natural product biosynthetic pathways in sequenced microbial genomes [9]. The biosynthetic potential of different strains isolated 413 ©Biomedical Informatics (2019) from various sources can be approximated by the detection of the genes involved in the synthesis of secondary metabolites such as those for a polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS). Non-ribosomal peptide synthetases (NRPSs) are megaenzymes usually with a multimodular structure, which catalyze the non-ribosomal assembly of peptides from proteinogenic and non-proteinogenic amino acids . Komaki and Harayama (2006) reported that DNA sequence based on these genes could be used to predict the chemical nature of compounds [14]. The biological functions of NRPS via synthesized compounds associated with the chemical nature of peptide, which is correlated with the gene sequence [11]. Therefore it is crucial to study the phylogenic insight of NRPS in the potential actinomycete would provide new opportunities for drug discovery.

Materials and Methods: Isolation and identification of Actinomycetes:
Soil samples were collected from Mangrove sediment of Pitchavaram (Latitude of 11.4' N-Longitude of 79.8' E), Tamil Nadu, India, in sterile airlock polythene bags and transported to the laboratory according to a previously described method [6]. One gram of air-dried each spot soil samples was added to a 9 ml of sterile water and subjected to selective pretreatment of dry heat at 56 °C for 10 min to effectively increase the number of myceliumforming actinomycetes relative to the non-actinomycetal heterotrophic microbial flora. After that, the samples were vigorously shaken and further diluted up to 10 -6 in sterile water. 100µl of each diluted sample was inoculated by spreading with a sterile glass rod onto humic acid-vitamin B agar (HV) medium (Hayakawa & Ohara,1987) supplemented with antibiotics of cycloheximide (40 µg/ml), nystatin (30 µg/ml) and nalidixic acid (10 µg/ml) after autoclave to inhibit the fungal and nonfilamentous bacterial growth. The inoculated plates were incubated at 30 °C for ten days or until appearance of colonies with a tough leathery texture, dry or folded appearance, and branching filaments with or without aerial mycelia.

Phylogenetic analysis of NRPS Adenylate domain
BLAST network services at the NCBI were used to analyze the resulting NRPSs gene sequence [16]. Multiple alignments were performed using CLUSTAL_X version 1.8 [17]. The phylogenetic tree was inferred Neighbor-joining method using MEGA 6.0 software package [18]. The unrooted phylogenetic tree topology was evaluated by using the bootstrap resamplings method with 1000 replicates [19].

Results and Discussion: Isolation and characterization of Mangrove Actinomycete:
The results of morphological, physiological and biochemical characteristics of strain BDUSMP 02 are shown in (Table 1). The cell wall of the strain found to contain LL-diaminopimelic acid (chemotype I), which is characteristic for the genus Streptomyces. Phylogenetic analysis of the 16S rRNA gene sequence (1388 bp) of strain BDUSMP 02 revealed that the isolate belongs to the genus Streptomyces. The 16S rDNA sequence has been deposited in the GenBank database under Accession No. KF918272.1. Based on morphological, physiological, biochemical characterization and 16S rDNA sequence analysis, the isolate was named as Streptomyces sp. BDUSMP 02.  In agreement with those previous reports, the results presented in this study denoted that bioactive secondary metabolite production by the mangrove sediment actinomycete and its gene clusters responsible for the biosynthesis could be at a later stage taken into the molecular biology of natural product research.

NRPSs gene adenylation domain (A-domain):
Streptomyces sp. BDUSMP 02 non-ribosomal peptide synthetase gene, partial cds, has been deposited in GenBank database under Accession No. KJ598809.1.The resulting amino acid sequences corresponding to their nucleotide sequences of amplified NRPS Adomain showed conserved motif, as shown in Figure 1. There are three core motifs in the amplified 450 bp fragments of A-domain identified including A2, TGxPKGV, A3, FD and A4, NxYGPTE.

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©Biomedical Informatics (2019) NRPS A -domain was best matched with the previously reported Streptomyces. The resulting amino acid sequences shared low similarities with those available in databanks. Liu et al. (2019) reported that Streptomyces isolated from mangrove sediment harbouring NRPS genes which involved in the synthesis of the antibacterial compound. Secondary metabolite production in Streptomyces is growth dependent and involves the expression of physically clustered regulatory and biosynthetic genes by a tightly regulated mechanism [21]. Similarly, in the present study, biosynthetic NRPS gene sequences provided valuable genomicbased information in parallel with the antimicrobial activity of isolate. Our results thus proved the presence of NRPS genes in support of the bioassay-guided analysis for antibacterial activity. Figure 2 presents the phylogenetic tree of Streptomyces sp BDUSMP 02 based on NRPS A-domain amino acid sequence. The NRPS Adomain amino acid sequence of the isolate Streptomyces sp BDUSMP 02 showed a less identity to the sequences from various Streptomyces sp. A good agreement between bioassay-guided identification antibacterial properties this isolate had functional NRPS genes in their putative gene cluster responsible for the synthesis of antibacterial compounds. Interestingly the strain NRPS A -domain shared similarity with Streptomyces avermilities. It is therefore desirable to isolate the secondary metabolite with antibacterial properties to relate with its functional genes.

Conclusion:
We describe a Streptomyces sp. from mangrove environment as a promising source of novel antibacterial compounds. There is increasing interest in the characterization of gene clusters, which mainly contain NRPS, PKS and NRPS/PKS in addition to culturedependent experimentation for distinct bioactivities. We found NRPS adenylate domain from the potential isolate, which can be further explored for the drug discovery using a genome mining approach.