Separation of PRN from bacterial media extract using TLC utilized various stationary phases such as silica gel G, GF₂₅₄, 60 F₂₅₄, KCI8 F, C18 Glass and several mobile phases. PRN can be detected on TLC under UV transilluminator and visualized by spraying diazotized sulfanilic acid (DSA) or Pauly’s, Ehrlich’s and van Urk’s reagent to develop maroon and violet color, respectively or H₂SO₄ on Silica Gel G plate. The R_f value for various TLC system served to identify PRN from different bacterial species. The compound has been analyzed by retention time in gradient HPLC system but isocratic solvent system of 45% water, 30% acetonitrile, and 25% methanol also separated pyrrolnitrin at 252 nm in preparative HPLC. Modifications in the polarity of solvents, mobile-stationary phase and elution methods are key strategies to quantify PRN using HPLC (Table 4). Yellow colored PRN molecule isolated from P. pyrrocinia absorbs at 252 nm with molar extinction coefficient of ε = 7500 in ethanol. Myxobacterial PRN also showed λ_max at 252 nm in methanol. Functional group stretching in FTIR vary with different PRN derivatives due to its structural features. Typical bond stretching at 1530 and 1375 cm⁻¹ characterized for nitro group while 3489 cm⁻¹ represent pyrrole ring. Similarly, PRN isolated from supernatant of fermented medium inoculated by Myxococcus fulvus strain Mx f147 indicated infrared spectrum to confirm pyrrole ring (3460), nitro group (1530 and 1375), CH₃ (stretch) (1460) and C=C aromatic weak intensity (1600). Mass spectroscopy (MS) of PRN is ascertain using different ionization techniques. MS of PRN isolated from Pseudomonas cepacia B37w showed molecular ion at m/z 256 with the formula C₁₀H₆C₁₂N₂O₂. Electrospray mass spectroscopy (negative ion spectrum) of PRN further confirmed (mass-to-charge ratio; m/z) at 256. High-resolution mass spectrometry of the two molecular ions gave m/z 255.9826 and 257.9777, respectively, indicating the molecular formula C₁₀H₆N₂O₂³⁵C₁₂ and C₁₀H₆N₂O₂³⁵Cl³⁷Cl.

Table 4. Several HPLC methods adopted to separate and quantify pyrrolnitrin from microbes using different solvent system.

Column	Flow Rate (mL/min−1)	Solvent System	Detector	Retention Time (min)
RP 18	2	Methanol: water (70:30; v/v)	-	-			-	Mol. Wt.: 192.64
-	ND	Antibiotic, antifungal nature		3-chloro-4(2-amino-3-chlorophenyl)-pyrrole	Di-chloro-amino (DCA) (amino-pyrrolnitrin)		212 (4.46)	302 (3.57)	C10H8Cl2N2	Exact Mass:
2.	P. aureofaciens, P. fluorescens, P. multivorans	-	226.01	CMM, Synthetic C, E		Mol. Wt.: 227.09
27 °C, shaker	7		0.32–126 (µg mL	−1
50 mm × 4.6 mm I.D. guard	1.0	)	Acetonitrile: methanol: water (1:1:1)	PRN widespread in groups of	Pseudomonas		UV (254 nm)	10		2, 3 dichloro-4-(2-amino-3-chlorophenyl)-pyrrole	Tri-chloro-amino (TCA)		212 (4.54)	302 (3.61)	C10H7Cl
3.	Silica gel (60 μm)	P. aureofaciens	3	-	-	Chloroform: hexane (1:1, 1.5:1, 2:1, 5:1) (v/v	CMM	N	); chloroform; chloroform-acetone (5:1, 1:1) (v	27 °C, shaker	2	/v	Exact Mass: 259.97 Mol. Wt.: 261.53
); acetone	Bioassay with	R. solani
Sephadex LH-20
Rainin Dynamax C18 (21.4 × 250 mm)	-	Acetonitrile: water (3:2; v/v	5	) fractions collected at 9.5 to 12.5 min and re-chromatographed on a silica column eluted with chloroform: hexane (1:1; v/v	9.5 to 50 (µg mL	−1)	Production of substituted PRN from Tryptophan analogs		)	-	13.5		3-chloro-4-(3-chloro-2nitro-phenyl)-1H pyrrole	Pyrrolnitrin (PRN)		212 (4.39)	252 (3.83)	C10H6Cl2
4.	N	2	P. aureofaciens	O	2	-	Exact Mass: 255.98	CMM	Mol. Wt.: 257.07
30 °C, shaker	5	18.35–19.9 (µm)	Possible pathway discussed		-	Methanol	pHPLC	2, 3 dichloro-4-(2-nitro-3-chlorophenyl) pyrrole	2-chloro-pyrrolnitrin (2-CPRN)		212 (4.47)	240
5.	Pseudomonas cepacia B37w (NRRL B-14858)	Rhizosphere	280	Sabouraud Maltose Broth	C10H	-	5Cl3N2O	Biocontrol property of plants protected from strain
Silica gel 60 (0.015–0.040 mm; Merck)	-
C-18 column, 5 µm	-	Isocratic acetonitrile: methanol: water (1:1:1)	-	-		2	Exact Mass: 289.94 Mol. Wt.: 291.52
6	2.133 (mg L	−1	)	Efficacy against	F. Sambucinum	incited potato dry rot disease		2-(2-Heptenyl)-3-methyl-4(1H) quinolone	-		-	-	C17H21
Hypersil octyldecyl saline (2.1 mm diameter by 10 cm)	6.	NO	Pseudomonas cepacia LT4-12- W	Apple leaves	Exact Mass:	Mineral Salt, Nutrient Broth, Kings medium B	27 °C, 200 rpm	255.16	7	Mol. Wt.: 255.35
1) MS: 51.50 (mg L	−1	) 2) NB: 7.20 (mg L	−1	) 3) KMB: 5.50 (mg L	−1	)	Production of phenylpyrrole metabolites with respect to time		2,3-dichloro-4-(2-nitrophenyl) pyrrole	Iso-pyrrolnitrin		-	-	C10H
7.	B. cepacian	6	-	Cl	2N2O2	Exact Mass: 255.98 Mol. Wt.: 257.07
Mineral Salt	27 °C, shaker	7	3-chloro-4-(2-nitro-3-chloro-6-hydroxyphenyl) pyrrole	Oxy-pyrrolnitrin		-	-	C10H6Cl2N2O3	Exact Mass: 271.98 Mol. Wt.: 273.07

Structurally, PRN possesses benzene and pyrrole rings with chlorine atoms on both of them and nitro and chlorine units to form an unusual natural skeleton. It has chlorine moiety to contribute more towards biological activity in comparison to its bromine derivative. Consequently, several natural congeners of PRN such as amino-pyrrolnitrin, iso-pyrrolnitrin, 2-chloropyrrolnitrin, oxy-pyrrolnitrin, 4-fluoropyrrolnitrin, and 3-fluoro-3-dechloropyrrolnitrin have been reported. Brominated derivatives of PRN can be synthesized by replacing chlorine ion with bromine in the presence of sodium bromide.

2.1. Pyrrolnitrin: Chemical Synthesis

PRN is positive towards Ehrlich’s reagent where pyrrole ring gets condensed with p-dimethylaminobenzaldehyde to form the violet color complex. Pauly’s coupling reaction yields red color and gives a negative reaction to the ferric chloride nitro group detection test. PRN can be oxidized by chromic acid to form corresponding compound which on oxidation with permanganate, yields carboxylic acid.

Modern synthetic targets for chemical synthesis require regiospecific polysubstituted aromatic or heteroaromatic components . PRN is chemically synthesized by α-block of pyrrole ring and 2-nitro-3-chloroacetophenone, and subsequent chlorination at 4-position and oxidation of methyles using sulfurilchloride followed by decarboxylation. In another approach, 2-Methyl-4-(2-nitro-3-chloro-phenyl)-5-ethoxycarbonyl-pyrrole was prepared in various steps from 2-amino-3-chloro-toluene. One of the most versatile synthetic approach for PRN allowed access to analog compounds such as monodechloroaminopyrrolnitrin and aminopyrrolnitrin. This step facilitated PRN synthesis using Suzuki–Miyaura cross-coupling of an appropriately halogenated pyrrole pinacolboronate ester with halogenated arylpyrroles using 2,6-disubstituted nitrobenzenes or 2,6-disubstituted anilines. Palladium-catalyzed coupling of 1-(triisopropylsilyl-3-substituted pyrroles with arylhaildes has also been described. However, chemical synthesis of PRN makes synthetic route cost extensive and pose threat to the environment. Furthermore, the chemical process utilizes noxious chemicals, high temperature and pressure, more energy and yield poor regioselectivity with lack of public acceptability. Thus, chemical industries prefer microbial species for more selective, greener and cost-effective approach for synthesizing PRN.

2.2. Microbial Pyrrolnitrin Production and Recovery

Microbial synthesis of PRN is easy, reliable and eco-friendly and requires low-cost medium constituents, ambient conditions for growth and production, the least additional energy requirements and minimum expensive equipment. This is the major reason microbial synthesis of PRN has become the preferred alternative to chemical processes. After initial isolation of PRN from Pseudomonas pyrrocinia and thereafter reports from different fluorescent and non-fluorescent Pseudomonas species, several strains of Burkholderia cepacia, Corallococcus exiguus, Cystobacter ferrugineus, Enterobacter agglomerans, Myxococcus fulvus, Serratia spp. and Actinosporangium vitaminophilum have been classified to produce PRN in varying quantities. Serratia plymuthica and

-

Dichloromethane then methanol

TLC

Water: methanol from 0%: 100 % and gradually changing up to 100%: 0%

-

between 10-15

Silica gel (H60)

-

Dichloromethane

Bioautography

ND

Delays postharvest fruit rot in strawberries

8.

Enterobacter agglomerans

Reverse phase 18

0.7

0 min 50% methanol in water

Silica gel

(20 × 170 mm, Wakogel C-200)

Benzene, 10% ethyl acetatobenzene, 20% ethyl acetate benzene and finally ethyl acetate

TLC

IC1270

Grapes rhizosphere

Potato Dextrose Agar

Incubated on agar plate

5

ND

Possible role of a combination of Chitinases and pyrrolnitrin in antagonism

Silicic acid

(240 × 22 mm)

Diethyl ether and methanol

-

15 min 100 % methanol in water

17 min 100% methanol in water 20 min 50% methanol in water 25 min 50% methanol in water

UV (252 nm)

15.4

C-18 reverse phase (125 × 4.6 mm)

-

Methanol: water (70:30; v/v)

UV (252 nm)

-

9.

-

RP C-18 flash

Water and methanol

TLC

-

1.0

2-min initialization at 10% ACN: 0.1% TFA; 20-min linear gradient to 100% ACN: 0.1% TFA

990-photodiode array detector

-

B. cepacia NB-1

Ponds in botanical garden of Tubingen, Germany

Minimal medium

27 °C, aeration rate 0·5 vvm, stirrer speed 150 rev min−1, pH −7.0

5

0.54 (mg L−1)

PRN block ETS Neurospora crassa 74 A; inhibition of Streptomycine spp.

10.

Burkholderia cepacia 5.5B (ATCC 55344) Wild Type

Soil sample, North Carolina

Nutrient broth, Mineral salt

25 °C, at 200 rpm, pH 5.8

5

NB: 35.59; MS: 28.54 (mg 1012 cfu)

-

RP C-18 (MPLC)

Biocontrol of

50% to 100% aq methanol

HPLC

Silica gel (60 μm)

-

Toluene

-

Nucleosil C-18

Acetonitrile: water (20 to 100%)

-

27.5

RP C-18 column

1.0

Rhizoctonia

Isocratically 45% water: 30% acetonitrile: 25% methanol

stem rot of poinsettia

-

-

11.

Pseudomonas fluorescens psd

Roots of Vigna mungo

C-18 RP column

10% acetonitrile: water (v/

Standard succinate medium (SSM)

v

-

-

) (both acidified with 0.1% amino acid) run for 2min linear gradient 100% acetonitrile (acidified with 0.1% amino acid)

-

18

ND

12.

Pseudomonas chlororaphis O6

-

Nutrient broth, Mung bean medium

28 °C 200 rpm

-

1.7 (µg mL−1)

Regulation by glucose of PRN production influenced biocontrol of tomato leaf blight

13.

Acinetobacter haemolyticus A19

Wheat rhizosphere

0.7

18 min linear gradient from 50 to 100% methanol and 0.1% trifluoracetic acid in methanol

-

-

NMR spectroscopy is widely used for analytical measurement of microbial metabolites. The PRN is confirmed by NMR spectrum with values: (i) 1H NMR: H-2 and H-5: 6.82 (m, 2H); H-5′: 7.41-7.53 (m, 3H); NH: 8.29 (br s, 1H); and (ii) 13C NMR δ value: 111.9 indicated C-3, 115.4 for C-4, 116.6 for C-5, 117.4 meant for C-2, while 124.8, 127.6, 128.6, 130.1, and 130.3 designated for C-3′, C-1′, C-6′, C-4′ and C-5′, respectively. Chemical shift (δ) values at 6.81 (m, 2H) indicate the presence of H-2 and H-5, 7.41: H-6′, 7.43: H-5′, 7.52: H-4′, 8.38: NH. NMR spectrum of purified PRN secreted by plasmid-mediated A. haemolyticus A19 revealed the values δ: 6.2–6.6 (m, 2H, H-2, H-5), 6.77 (q 1H, H6), 7.03 (m, 1H, H-4), 7.38 (m2H, Ha, Hc) compared with standard 1H NMR spectrum of. PRN synthesized from Myxococcus fulvus strain Mx f147 showed 13C NMR spectrum (in acetone-d6; Bruker 400 MHz). Structural investigation of PRN with X-ray analysis revealed the presence of two molecules with observed density of 1.74 g/cm³ that lie opposite to each other about the center of symmetry. It further confirmed the location of two Cl atoms in the asymmetric unit with 3D Patterson function, dihedral angle of the pyrrole, the benzene rings and chlorine substitution on pyrrole ring located apart from the nitro group.

2.4. Biochemistry of Pyrrolnitrin

Microbial synthesis of PRN requires D-tryptophan, but cost of precursor amino acid and intracellular secretion limits its large-scale production. The NO₂ group is derived from anthranilic acid, phenylalanine and tryptophan that could serve as a precursor for PRN secretion. However, anthranilic acid and L-phenylalanine usually decrease PRN secretion in P. aureofaciens and B. cepacian, while tryptophan stimulates PRN production. In the medium, L-tryptophan gets quick intake within the cells than the D- isomer but addition of L-isomer could not yield more PRN secretion. In actinomycetes, D-tryptophan enhances secretion of PRN when added separately in the culture medium and maximum accumulation was observed at stationary phase after 120 h. It indicated that the L-isomer of tryptophan enter cells quickly and participate in the protein synthesis, while D-tryptophan enter slowly and available at the time of antibiotic secretion. Besides, L-glutamic acid amended medium showed maximum antifungal activity, which substantially declined with the addition of L-tryptophan, L-valine, L-serine, L-phenylalanine and L-cysteine. In brief, D-tryptophan and L-glutamic acid are more direct precursors of PRN than any other amino acids.

PRN biosynthesis was unraveled in P. aureofaciens. Later, genes (prnABCD operon) and corresponding enzymes involved were delineated in P. fluorescens BL915 (Figure 2).  The biosynthesis of PRN occurs in four sequential steps: chlorination by prnA, rearrangement and decarboxylation by prnB, chlorination by prnC and oxidation by prnD enzyme (Figure 1). This involves regioselective halogenation of tryptophan through the addition of chlorine into D-tryptophan by tryptophan 7-halogenase (prnA) following nucleophilic and electrophilic reactions and activation of intermediate lysine-chloramine species as the first step. Further, the reaction catalyzed by prnB shows structural similarity with two-domain indoleamine 2,3-dioxygenase enzyme (IDO) and involves several intermediary steps. The second step forms a binary complex that combines with L-tryptophan or 7-Cl-L-tryptophan to create a ternary complex. The third step in PRN biosynthetic pathway of P. fluorescens leads to catalytic conversion of mono-chloro-deamino-pyrrolnitrin into amino-pyrrolnitrin by regioselectivity using halogenating and chlorinating enzyme. In the last step, prnD catalyzes the oxidation of amino group of aminopyrrolnitrin to nitro group and thus forms PRN.

Figure 2. Cluster organization of pyrrolnitrin biosynthetic genes in S. plymuthica PR1-2C (1), P. protegens Pf5 (2), B. pyrrocinia (3) and myxobacterium Melittangium boletus (4). Nucleotide sequence (NT size indicated for prnA–D genes) of the bacterial species was obtained from KEGG database.

Aminopyrrolnitrin oxidase or arylamine oxygenase (rieske N-oxygenase) catalyzes oxidation of an arylamine into the arylnitro group. Except prnB, trptophan-7-halogenase (prnA), monodechloroaminopyrrolnitrin (prnC) and aminopyrrolnitrin oxidase (prnD) enzymes require flavin reductase (prnF) gene located close to the prnABCD operon which is considered as a part of the cluster. Bioinformatics clubbed with the biochemical tools identified the role of prnF gene in prnD-catalyzed unusual arylamine oxidation in P. fluorescens Pf-5. The prnF and prnD genes form a two-component oxygenase system, in which the gene product enzyme prnF supplies the reduced flavin to prnD. The prnF requires NADH as an electron donor to reduce FAD so that reduced FAD supplies electrons from NADPH to the prnD oxygenase component through protein-protein interactions in order to protect the flavin from oxidation.

The prnF gene having molecular mass of 17kD with GC content of 62%, encodes for a polypeptide chain of 160 amino acids. The enzyme belongs to flavin:NAD(P)H reductases family with part of two-component monooxygenase systems and its C-terminal region possesses highly conserved GDH motif for NAD(P)H binding]. It resembles with PheA2, SnaC, VlmR, ActVB and HpaC with 31.5%, 28.6%, 26.4%, 25.6%, and 25.5% amino acid identity, respectively.

2.5. Pyrrolnitrin Derivatives

Several halogen variations of the PRN molecule have been isolated in the past in the form of bromo-analogs of pyrrolnitrin from fermentation of P. aureofaciens in sodium bromide with low antifungal activity. In addition, 2-chloropyrrolnitrin contain an additional chlorine atom which possesses about 10% of the antifungal activity of PRN. The pyrrolomycin (B, C, D, E, F₁, F_2a, F_2b, and F₃) derivatives encompass a chlorine or bromine atom at the 3-position of the pyrrole ring, and either two chlorine atoms at positions 4 and 5 or one chlorine and one bromine at any of these positions have shown significant antifungal activity. Novel oxidized derivatives of pyrrolnitrin including two new pyrrolnitrin analogs, namely 3-chloro-4-(3-chloro-2-nitrophenyl)-5-methoxy-3-pyrrolin-2-oneand4-chloro-3-(3-chloro-2-nitrophenyl)-5-methoxy-3-pyrrolin-2-onehave, were reported from B. cepacia K87.

Furthermore, number of de-chloro and de-nitro derivatives of PRN and the isomers were synthesized by cyclization of enamine reaction, hydrolysis, carboxylation and Mannich’s reaction. The strongest antifungal activity of PRN and its analogs resulted when it got unsubstituted by ester group at any position. The antifungal activity become more stronger when shift of NO₂ group was increased. Few PRN derivatives such as denitropyrrolnitrin (3-chloro-4-(3-chlorophenyl), bromo analog: 3-chloro-4-(3-bromophenyl)pyrrole) and trifluoromethyl derivative (3-chloro-4-(3-trifiuorornethyl) pyrrole) were strong antimicrobials. However, among all the analogs homologous to NO₂ group of pyrrolnitrin, PRN has remained the strongest biologically active compound. The UV irradiation of 2-(pyrrol-3-yl)nitrobenzene moiety of PRN in an anhydrous aprotic solvent yielded 7,4′-dichlorospiro(1,3-dihydrobenzo(c)isoxazole-3,3′-pyrrolin-2′-one) by the intramolecular oxidation. Hence, the photodegradation of PRN depends on aqueous reaction media and the nature of its excited state.

3. Applications of Pyrrolnitrin

3.1. Biological Activity

Structure–activity mechanism reveals that the primary target of PRN lies in the cell membrane to impede protein, RNA, DNA synthesis and uncouple the normal electron flow in the respiratory electron transport chain. The metabolite has demonstrated biological activity at low concentration and act as an uncoupler of oxidative phosphorylation in N.eurospora crassa. High concentration of PRN causes impairment of electron transport in flavin region and cytochrome c oxidase; accumulation of glycerol; synthesis of triacyl glycerol leading to leakage of cell membrane and inhibition of cell growth; in vitro activity against bacteria and fungi in the range of 1–100 µg mL⁻¹; in vitro activity against leukemia and melanoma cell lines; and moderate antimycobacterial activity at 8 µg mL⁻¹. The halometabolite was used as a drug lead for fenipoclonil and fludioxonil synthesis. The amino derivative of PRN was identified as an androgen receptor antagonist. PRN has the unique property to persist actively in the soil over a month, and can be readily diffused and slowly released after lysis of host bacterial cell. However, the compound is sensitive to decomposition due to light.

Inhibitory effect of PRN is seen on the mitochondrial electron transport system of Neurospora crassa 74A. Studies using N,N,N’,N’-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) confirmed that PRN block transfer of electron between the dehydrogenases and cytochrome c-oxidase components of the respiratory chain. At low concentrations, PRN uncouples oxidative phosphorylation in Neurospora mitochondria and impedes electron transport in both the Flavin region and cytochrome C oxidase at high concentration. PRN also function as a signal molecule, beyond its role as a bioactive molecule to suppress fungal and affected cell motility. Antifungal activity of the compound increased at pH 6.0, became maximum at pH 10 or 11 and declined after pH 11. Temperature influence on antifungal activity was maximum at 28 °C. Similarly, 2% NaCl content in the medium showed maximum activity. Such studies indicated more scope for medium modifications for obtaining maximum PRN production followed by maximizing biological activity of the compound.

The quorum-sensing system related regulation of PRN is reported in a chitinolytic bacterium S. plymuthica strain HRO-C48, that protects oilseed rape crop from Verticillium wilt. The mutant deficient in PRN production shown the ability to produce the compound in the medium supplemented with chemically synthesized N-hexanoyl-HSL and N-3-oxo-hexanoyl-HSL (OHHL) (100 µM), thus suggesting that quorum sensing (QS) ability regulated PRN biosynthesis. While investigating the role of N-acylhomoserine lactone (AHL)-dependent quorum sensing for expressing antifungal traits, It was found that PRN expression was positively regulated by CepR gene at transcription level. PRN is reported to have significant antimicrobial potential (Table 5) against Streptomyces antibioticus, S. violaceoruber, Paecilomyces variotii and Penicillium puberulum. However, S. prasinus, S. ramulosus, Aspergillus proliferans and A. terreus showed tolerance to pyrrolnitrin. PRN displayed activity against Ustilago maydis, Candida albicans, Hansenula anomala, Arthrobacter oxidans, Bacillus coagulans, B. lichenifernis, B. subtilis and B. thuriengiensis at low concentration. Pyrrolnitrin produced by Pseudomonas chlororaphis strain PA23 exhibited nematicidal and repellent activity against Caenorhabditis elegans. Co-culturing P. chlororaphis and C. elegans enhanced expression of biocontrol-related phzA, hcnA, phzR, phzl, rpoS and gacS genes and, thus, contributed to the fast killing of nematode in bacterial interaction.

Table 5. Bioactivity spectrum of pyrrolnitrin against bacteria, fungi and nematodes.

Sr. No.	Name of Test Microorganism	PRN (µg mL−1)
Bacteria
1.	Staphylococcus aureus 209-P	6.2
2.	Escherichia coli	250
3.	M. tuberculosis CIP 103471	4.0
4.	M. avium CIP 103317	8.0
5.	M. smegmatis CIP 103599	16.0
6.	M. gordonae CIP 6427	>16.0
7.	M. marinum CIP 6423	>16.0
Yeast
8.	Candida albicans	1.0
9.	Saccharomyces cerevisiae	10.0
10.	Cryptococcus neoformans	< 0.78
11.	Candida albicans	12.5	Luria broth	-	2	15 (mg L−1)	Plasmid-mediated pyrrolnitrin production by A. Haemolyticus A19
14.	Pseudomonas chlororaphis strain PA23	-	M9 medium + 1 mm MgSO4 + 0.2% glucose	-	5	ND	Nematicidal and repellent activity against Caenorhabditis elegans
15.	Serratia marcescens ETR17	Tea rhizosphere	Semi-solid pigment producing media	30 °C	8	ND	Effective reduction of root-rot disease tea plant on talc-based formulations; Plant growth promoting activity

CMM: Citrate minimal medium; ND: not determined; NB: Nutrient broth; MS; Murashige-Skoog medium; cfu: colony forming units; KMB: King’s medium-B.

Besides intracellular production of PRN from Pseudomonas spp., the excretion of the compound was also detected in the supernatant of fermented medium of S. marcescens strain ETR17. B. cepacia yielded 0.54 mg L⁻¹ of PRN in monosodium glutamate medium at 27 °C as quantified by preparative HPLC. Earlier it was reported that only 27.58% Pseudomonas spp. secreted PRN in shake flask fermentation propagated in CMM, C, or E media. The authors concluded that P. multivorans C653 (ATCC 17760) showed maximum PRN production in medium C, followed by E and then CMM. P. aureofaciens was shown to secrete moderate PRN in CMM medium (40–80 µg mL⁻¹). The PRN concentration increased in D-tryptophan amended medium where it was incorporated in the biosynthesis of PRN.

While growing P. aureofaciens in isotopically labeled tryptophan (at different positions) containing medium, It was demonstrated that amino nitrogen of D-tryptophan became the nitro group of PRN. The two chlorine atoms in PRN, C3 of side chain became pyrrole and C2 of the indole nucleus got retained during biosynthesis (Figure 1). Furthermore, it was confirmed that H-2 and H-α of the indole and side chain give rise to H-5 and H-2 of PRN, respectively, and, thus, proposed that L-tryptophan is the immediate precursor in PRN biosynthetic pathway. PRN formation using labeled tryptophan showed that L- rather than D-tryptophan was the immediate precursor of PRN. 7-chloroindole-3-acetic acid, 3-chloroanthranilate detected in fermented medium revealed that 7-chlorotryptophan served as a common precursor for PRN.

Variety of production media and their pH remained a key parameter to influence PRN secretion. Shake flask fermentation of P. cepacia LT4-12-W revealed that the final yield (at 168 h) of PRN almost doubled at pH 5.8. Amendment of MS medium with glutamate salt of sodium yielded 60.50 mg mL⁻¹ of PRM secretion. The effect of different physicochemical conditions on plasmid-mediated PRN secretion has also been reported from Acinetobacter haemolyticus A19 isolate from wheat rhizosphere.

Recovery strategy of PRN involves cell growth in appropriate medium, extraction in acetone followed by removal of oily matter from concentrated acetone solution using petroleum benzene. From fermented broth at pH 10 or 11 (6 mL) with NaOH, cell pellet centrifugation following sonication with acetone (600 µL) for 1 min, separation of acetone supernatant, re-extraction of pellets again in acetone (300 µL) and drying of acetone extract also yield PRN extract. Further, fermented cultures were extracted after 48 h with equal volume of ethyl acetate and centrifuged. Pellets sonicated twice with ethyl acetate (5 mL) for 3 min then recovery of organic phase resulted in PRN rich dried extract. Lysis of 18 h culture of A. haemolyticus A19 using 1% SDS followed by sonication for 5–15 min resulted in supernatant with PRN. In the case of bioactivity and characterization study, chromatographic separation techniques such as column chromatography and flash column with different mobile phases were explored (Table 3).

Table 3. Purification of pyrrolnitrin using various separation techniques with different solvent systems.

Matrix	Column

TLC: thin layer chromatography; HPLC: high performance liquid chromatography; Aq.: Aqueous; MPLC: medium pressure liquid chromatography; RP: Reverse phase; pHPLC: preparative HPLC.

-
-
30 ~ 60% aq acetonitrile (for 70 min)
-
68.9
Gemini C18 column (100 × 4.6 mm; 5mm particle diameter)	1.0	Isocratically 45% acetonitrile: 35% water: 20% methanol	Dionex AD20 (Dionex,Sunnyvale, CA) (225 nm)	-
	Cosmosil C18
12.	Cryptococcus neoformans	0.78
13.	Candida utilis	10.0	]
Fungi
14.	Trichophyton asteroids	0.05
15.	Sporotrichum schenckii	< 0.78
16.	Penicillium atrovenetwn	10.0
17.	P. oxalicwn	10.0
18.	Sporotrichum schenckii	3.12
19.	Blastomyces dermatitidis	< 0.78
20.	Histoplasma capsulatu	0.156
21.	Sclerotinia sclerotiorum	0.01
22.	Rhizoctonia solani	50 (µg/disc)
Nematode
23.	Caenorhabditis elegans	0.1

Bacterial growth inhibition by PRN forms complex with phospholipids of cell membranes that eventually cease cellular respiration. Furthermore, PRN causes leakage of A260 mµ absorbing material inside the cells and impairs synthesis of protein, DNA and RNA. However, in vitro protein synthesis in PRN treated Rhizoctonia solani and Escherichia coli remained unaffected. It bursts protoplast of B.acillus megaterium KM at growth inhibitory concentration. The multitudes and range of activity of PRN makes it a preferred bioactive compound for agricultural chemical sector.

3.2. Agricultural Applications

Phenylpyrroles were proven and effective agents against Trichophyton, Microsporium, Epidermophyton, Penicillium, Candida spp. and several Gram-positive bacteria. Besides, PRN showed activity against soilborne fungal phytopathogens Rhizoctonia solani and Fusarium sambucinum and foliar fungal pathogens Fusarium graminearum, F. culmorum, Pyrenophora triticirepentis, Thielaviopsis brasicola, Verticillium dahlia and Alternaria spp. The compound inhibited Gaeumannomyces graminis of wheat, Alternaria brassicae and Botrytis cinerea, and partially inhibited Fusarium roseum. Remarkable inhibition of mycelial growth and conidial germination was observed at a PRN concentration of 0.4 µg mL⁻¹ compared to phenazine-1-carboxylic acid at 50 μg mL⁻¹. Results suggest strong possibility of the compound being a prospective biocontrol agent in the agriculture.

The fungistatic effect of PRN was most distinct against Alternaria sp., Botrytis cinerea, Pythium aphanidermatum, P. ultimum, Rhizoctonia solani, Rhizopus sp. Aspergillus niger, Fusarium oxysporum, Penicillium expansum and Sclerotium rolfsii. Antibacterial activity was also recorded against Agrobacterium tumefaciens, Corynebacterium insidiousum, Pseudomonas syringae pathovar syringae, and Xanthomonas campestris (Minimum Inhibitory Concentration (MIC) ≥1 μg mL⁻¹). Organisms such as Clavibacterium michiganense and S. marcescens were suppressed at MIC ≥ 10 μg mL⁻¹. Moderate activity against Gram-positive and Gram-negative bacteria was seen at 12.5–100 mg mL⁻¹ (MIC). Strong toxicity was noticed against fungi, especially trichophytes, Trichophyton asteroids, at MIC of 0.05 mg mL⁻¹. In addition, PRN as a nitro-heterocyclic chemotherapeutic agent exhibited antimycobacterial activity against M. tuberculosis and M. avium. At present, only two synthesized derivatives of PRN, namely fludioxonil and fenpiclonil analogs, were registered as agricultural fungicides in France and Switzerland, respectively. Commercial products of fenpiclonil and fludioxonil include BERET, GALBAS and SAPHIRE, CELEST and MAXIM sold by Syngenta, respectively.

PRN found most prolific applications in controlling damping-off disease of cotton and cucumber, tan spot of wheat, storage molds of pome fruits, seedling disease of cotton, dry rot of potato and sclerotinia wilt of sunflower. More usage of the compound lies in its significant antibiotic activity and low toxicity to mammalian species. Wounds on apple and pear were challenged with a conidial suspension of antagonist grey mold B. cinerea and blue mold Penicillium expansum to investigate the efficacy of pyrrolnitrin (6–200 µg mL⁻¹) to control diseases at 2 and 24 °C after harvest. High concentrations of PRN proved effective at 24 °C on both diseases of apple and pear, while low concentrations appeared effective at cold temperature. Hence, PRN is an attractive strategy to control postharvest diseases on fruits, vegetables and other agricultural products being produced at low temperature conditions. In a preliminary field experiment on strawberries, postharvest treatment with PRN (250 mg L⁻¹) at low storage temperature delayed development of post-harvest rot by 2–4 days, but did not reduce rate of development and spoilage to acceptable levels.

In greenhouse studies, PRN showed prominent activity against Pyricularia oryzae and Botrytis cinerea. The PRN producer P. chlororaphis O6 has shown antifungal activities both in vitro and in planta on tomato against late blight disease and demonstrated major antagonism. In addition, biocontrol of fungal disease Fusarium Head Blight (FHB) caused by F. graminearum on wheat heads in growth chamber conditions was studied using strain P. chlororaphis G05 co-treated with: (i) wild-type strain G05; (ii) phz-deleted mutant G05Δphz; and (iii) mutant G05Δprn. The experiment showed wheat heads were infected with F. graminearum at rates of 5–8% and 80–90%, respectively, when co-sprayed with wild-type strain G05 and mutant G05Δprn, and PRN of wild type strain was found to be vigorously active against FHB disease.

The glasshouse experiments with talc-based formulation of S. marcescens ETR17 were similar to in vitro studies. Incidence of root rot in bacteria treated tea plants were considerably lower in comparison to untreated control as well as the fungicide treated sets. Additionally, ETR17 formulation also increased the root and shoot length of the tea seedlings under both sterile and unsterile soil conditions in comparison to the untreated controls.

3.3. Pharmaceutical Applications

Pyrrolnitrin demonstrated strong protecting activity against various pathogenic fungi, especially against dermatophytosis. It has been recommended for the treatment of superficial fungal infection of dermatophytic Trychophyton in Japan. A patent has been granted on antifungal composition containing pyrrolnitrin and antimycotic imidazole compound in 1987. The product was commercialized under trade name Pyro-Ace W powder Spray by Fujisawa Pharmaceutical Company Ltd., Osaka. This was marketed by Pharmacia in Italy as “Micutrin” and, in combination with betamethasone valerate, it was formulated as “Beta Micutrin” for athlete’s foot and ring worm diseases. The derivative, 3-cyanopyrroles, is more biologically active as pyrrolnitrin and very stable under light. Jespers and co-workers (1993) reported a Fenpiclonil (CGA 142705) with more cytotoxicity for the representatives of Ascomycetes, Basidiomycetes, and Deuteromycetes. PRN formulated with carboxymethyl cellulose (5%) was injected intraperitoneally into mice and LD₅₀ was observed at a dosage of 500 mg Kg⁻¹.

In pharmacology, in vitro radioactive studies of pyrrolnitrin reflected that pyrrole ring is readily oxidized by enzymes undetected in urine and bile after administration. Along with this, surface antigens of Candida albicans were released after treatment with PRN. It also showed cytotoxicity at 10 µg mL⁻¹ after 24 h and highest after 72 h on rat clonal pancreatic β-lines, INS-1. Thus, the compound becomes diabetogenic but appears nontoxic and insulinotropic at lower concentration. PRN affected physiology of Caenorhabditis elegans, acted as repellent for adult nematodes to lower egg hatching by almost <50% at higher concentrations of PRN (1, 5, and 10 μg mL⁻¹) after 24 h of exposure.

The review article and the statements therein, is Referenced as per the citations available with the MDPI Biomolecule original MS published in Biomolecules in 2020.  

Authors : Shraddha P., Chaudhary A. L., Prabha, Ratna, Renu, S., Singh, Dhananjaya P.

Submitted by : Dhananjaya P. Singh