The secretion of PRN in soil by few rhizospheric bacteria has contributed potential role in the (i) suppression of soil to plant pathogens and, (ii) organically driven agricultural practices (Costa et al. 2009). Rhizospheric RJ soil appeared strong brown coloured, with intense dried cracks, while RD soil seems to be dark red to black in colour, possibly due to high iron content. The alkalinity and salinity probably served as key determinants for the occurrence of rhizobacteria in soil to produce PRN (Garbeva et al. 2004) and hence, rhizobacteria application to crop has emerged out as preferred eco-friendly strategy to control phytopathogens compared to the synthetic chemical fungicides.
The rhizobacteria belonging to the genus Serratia was found in root exudates of plants that had affinity towards tryptophan. The root exo-metabolite contains tryptophan in detectable amount in radish root exudates, but, the in vivo concentration of PRN could be produced from available tryptophan is within the range required for fungal inhibition. Hence, secretion of PRN is always in limited quantity than other bacterial metabolites. Interestingly, the previous report had shown that bacterial cell retains PRN inside the cell more than exterior (Roitman et al. 1990) and hence, in vitro antibiosis was examined with supernatant to select efficient rhizobacteria against a test phytopathogen F. oxysporum. Antagonistic activity towards targeted phytopathogen was demonstrated in 23.42% bacterial culture extracts. The significant suppression of fungal development was seen with cell free broth, indicating extracellular secretion of antibiotics by rhizobacterial isolates. About, 72.22% Serratia species had displayed antifungal activity due to PRN secretion (Kalbe et al. 1996). Serratia marcescens (Kim et al. 2008) also, IC1270 and IC14 of S. plymuthica strains reported antagonistic activity due to secretion of PRN (Meziane et al. 2006; Liu et al. 2007), indicating that present study is in line with earlier reports.
The presence of pyrrole ring in the cell free broth was detected using Ehrlich reagent (2% p-dimethyl-amino-benzaldehyde), where reaction occurs with available α-hydrogen of pyrrole in boron trifluoride methanolic complex (Mattocks 1967). Ehrlich reagent in ethanolic hydrochloride formed weak and unstable colour complex that fade instantly during TLC analysis. Of 111 rhizobacterial isolates, only solvent extract of 15 bacterial strains demonstrated the presence of pyrrole ring and nitro group using FTIR and HPLC. The analytical tools revealed PRN secretion only in 13.5% rhizobacteria isolates possibly due to (i) undetectable secretion and (ii) absence of metabolic system to synthesize the compound from tryptophan. PRN from microbes was detected only in D-tryptophan amended MS medium and (El-Banna and Winkelmann 1998) indicated L-tryptophan had no effect on PRN production possibly tryptophan enters the cell quickly and utilized for protein synthesis. The initial characterization with UV, TLC, Ehrlich’s reaction test, IR and HPLC of metabolites secreted from various genera of bacteria demonstrated that the secretion of PRN is not restricted to Pseudomonas or Burkholderia. The chromatography (GC and HPLC) and mass spectroscopy (LCMS and GCMS) confirmed extracellular secretion of PRN from S. marcescens TW-3 and S. nematodiphila TO-2. PRN was isolated from bacterial cell extract (Arima et al. 1964; de Souza and Raaijmakers 2003) but, extracellular PRN was recorded up to minimum 5% secretion as contrast to the Roitman et al. (1990) who reported 98% phenylpyrrole present in the cell extract while only 1% found in the supernatant. B. cepacia in monosodium glutamate medium at 27 0C for 5 days showed very low yield of PRN (0.54 µgml− 1) by intracellular secretion (El-Banna and Winkelmann 1998). These findings demonstrated that PRN production is not unique to the genus Pseudomonas and our study uncovers PRN production by newer rhizospheric bacteria possibly contributing to selective antagonistic activity against fungal phytopathogens (Garbeva et al. 2004).
The preliminary morphology and 16S rRNA sequencing remarkably showed prevalence of rhizobacteria that are still unknown for the presence of phenyl-pyrrole/PRN-producers. Hence, prnD was used as a marker gene for screening of PRN producer strains. For this, five bacterial isolates screened initially for the PRN were detected for the presence of prnD with specific primers (PRND1 and PRND2) developed from conserved sequences (de Souza and Raaijmakers 2003). PCR amplification of prnD in three isolates namely, S. rhizophila KMB, Enterobacter spp. M-2 and B. parabrevis M-11 revealed absence of a distinct band, suggesting unsuccessful amplification of prnD gene (Fig. 6c). It may be possibly due to (i) dissimilarity of prnD gene sequences from that of Pseudomonas spp. or may harbour distant prnD gene and (ii) mismatches between the primers used and DNA template of respective strain i.e. primers specificity probably restricted to Pseudomonas and Burkholderia spp. only (de Souza and Raaijmakers 2003; Garbeva et al. 2004; Costa et al. 2009) and necessitate to optimize PCR or RT PCR with new specific primers for the occurrence of prnD coding genes in rhizobacteria KMB, M-2 and M-11 strains. These results agree with earlier report (Costa et al. 2009) where two Serratia strains 5.1R and 5.3R were not amplified with prnD. In the present study, both isolates TO-2 and TW-3 have demonstrated PRN production with chemical tools and, shown distinct band after PCR amplification but, the gene sequences (KY867430 and KY867431) (Fig. S1B) were not matching with prnD gene after BLASTn analysis. Although, rhizobacteria TO-2 and TW-3 have shown to secrete PRN by analytical tools but undetected for prnD may be due to polymorphic nature of prnD sequences (Costa et al. 2009). However, these prn gene sequence have shown 99% similarity with putative NADPH dependent FMN reductases present in complete genome of S. marcescens after BLASTn. Additionally, BLASTx revealed that the gene sequences belong to FMN reductase superfamily and thus, suggest the presence of prn gene which possibly codes for the supply of reduced flavin during enzymatic reactions.
The earlier reports suggested the role of FAD dependent prnF to supply the reduced flavin for functioning of prnD as product in PRN secretion (Hammer et al. 1997; Tiwari et al. 2012). However, functional or structural differences between the FMN-dependent and FAD-dependent enzyme systems were undetected (Ellis 2010). Hence, the query sequences (KY867430 and KY867431) in the present study were considered as prnF gene. On the contrary, earlier study had shown that prnF is non-specific and not directly involved in PRN biosynthesis (van Pee 2012) but, it is required for prnD to function and enhance PrnDs activity (Hammer et al. 1997; Lee et al. 2005; van Pee 2012). The bacterial DNA lacking prnF but having prnD gene may lose their ability to form PRN and thereof accumulate aminopyrrolnitrin (van Pee 2012). Aminopyrrolnitrin oxygenase (prnD) catalyses unusual arylamine oxidation which is only characterized example of arylamine N-oxygenases involved in arylnitro group formation (Lee and Zhao 2007) As stated earlier, monooxygenases are the flavoprotein enzyme that carries two reactions on a single polypeptide chain. The reduction of flavins followed by the transfer of reduced flavins to the oxygenase component by flavin-dependent reductases is the common reaction observed in two-component oxygenase family. Reduced form of flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and riboflavin by NAD(P)H are required to activate oxygen by the terminal oxygenases. However, the earlier study suggests reductase involved in two-component oxygenase of Pseudomonas fluorescens Pf-5 prefers FAD specific for NADH (Hammer et al. 1997) In addition, it has shown that the purified PrnD of P. fluorescens Pf-5 requires NADPH, FMN and flavin reductases for its functional expression in in vitro. The expression of prnF with prnD showed 15-fold higher activity of prnD than that of E. coli BL21 carrying only prnD (Lee et al. 2005). Moreover, single gene encoding an FMN reductase (prnF) express in heterologous hosts may not predict the role in PRN secretion. It necessitates coexpression with prnD and to this, requires (i) specific primers developed from conserved sequences of Serratia spp. for prnD, (ii) amplification of fragments with Serratia that produce PRN and, (iii) gene loci and its regulation in Serratia spp. The present study provided distinct reaction of PRN biosynthesis where NADPH dependent FMN reductase is involved to form reduced flavin in Serratia spp. FMN-dependent monooxygenases have been characterized in antibiotic synthesis in several Streptomyces spp. (Thibaut et al. 1995; Filisetti et al. 2003; Volton et al. 2008). As per the earlier reports, (i) reductases from the two-component system is specific for FMN, but monooxygenase may utilize either FMN or FAD also, (ii) FMN reductase differed in their specificity for NADH and NADPH (Sucharitakul et al. 2005; Ellis 2010) supporting to NADPH dependent FMN in single reaction for PRN secretion. This study signifies the role of PrnF in PRN biosynthesis (Tiwari et al. 2012)
The functional characterization of a studied protein sequence was facilitated by developing three-dimensional (3D) structure of protein using comparative or homology modelling which provided a structure related to one known protein. PrnF is superimposed with reductase part of the Escherichia coli K12 (3SVL). The objective to perform molecular docking is to (i) gain optimized conformation of PrnF, FMN and NADPH i.e. ligand-receptor complex with less binding energy and (ii) predict binding parameters of ligand-protein complex. The docking simulation with modelled PrnF has shown that several amino acids are in close contacts with the ligands i.e. FMN and NADPH (Fig. 7c). Overall, FMN is bounded with protein through hydrogen bond along with van der Waals, carbon-hydrogen, Pi-Sigma and alkyl bonds (Fig. S3b). The isoalloxazine ring of FMN probably fixed in the deep groove of the protein whereas NADPH restricted at wide groove with compact bonded conformation with nicotinamide ring where, binding capacity of protein-NADPH and protein FMN was − 5.7 and − 5.0, respectively.
Theoretical indications of the PrnF reductase in the two-component aryl amine oxygenase system allows to continue more detailed investigation of this system by genetical-structural characterization with protein engineering and interaction studies. Also, PRN protein model suggests highly restricted conformational space in active site and necessitate to relate the gene with various physiological scenario, interaction of biomolecules with enzymes. These comparative models provide the value of domains/proteins belonging to Serratia spp. in the stereoselective synthesis of pharmaceutically important drugs (Peng et al. 2014; Bai et al. 2015; Liu et al. 2018). In conclusion, preliminary molecular modelling analysis represents the basic study on prnF and further detailing of prnF with FMN and NADPH may pave more insight on two component arylamine oxygenase system in PRN production among rhizobacteria.