Mikrobiol. Z. 2021; 83(3):14-23.
doi: https://doi.org/10.15407/microbiolj83.03.014

Ecological Aspect of Antibiotic Batumin Synthesis by Pseudomonas batumici

V.V. Klochko1,2, I.I. Lipova1,2, N.V. Chuiko1, L.V. Avdeeva1

1Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Akad. Zabolotny Str., Kyiv, 03143, Ukraine

2National Technical University of Ukraine «Sikorsky Kyiv Polytechnic Institute»
37 Prosp. Peremohy, Kyiv, 03056, Ukraine

The species Pseudomonas batumici, isolated from the rhizosphere of eucalyptus in the humid subtropical zone, is a producer of the polyketide antibiotic batumin with highly selective activity against staphylococci. Batumin biosynthesis operon includes 28 genes or 74 151 bp. According to modern notions, the biosynthesis of energy-intensive metabolites, which probably includes batumin, is justified in the case of its multifunctionality for producers. The species P. batumici, as a representative of rhizosphere bacteria, must interact with plants and compete with the surrounding microbiota. Aim. To determine the role of batumin in the ecology of the rhizosphere producer strain P. batumici UCM B-321. Methods. The batumin producing strain P. batumici UCM B-321T was obtained from the Ukrainian Collection of Microorganisms. Antibiotic batumin was obtained by fermentation of P. batumici UCM B-321. Extraction was carried out from acidified P. batumici fermentation broth by chloroform (1:2). Chromatographic analysis of fermentation broth obtained after centrifugation was carried out by HPLC using liquid chromatograph Agilent 1200 with mass spectrometric detector Agilent G1956B. Batumin derivatives were obtained after the extraction of the fermentation broth of P. batumici using thin layer chromatography (TLC) on silica gel plates (Merck, USA) in the benzene-isopropanol system (5:1). Disc-diffusion method on phytopathogenic test-strains was used for bioautography. Biofilm formation by P. batumici strain was studied according to O’Toole by growing strain B-321 at 25 0C for 48 hours in 96-well plates on LB medium. Batumin effect upon bacterial mobility was studied using Volf and Berg method in Petri dishes with 0.5% semisolid bacterial agar. To research chemotaxis the soil strain Bacillus subtilis IMV B-7023 and the following concentrations of batumin were used: 20, 50, and 150 μg/mL. The studies were performed using Tso and Adler method. Results. Growth inhibition zones for phytopathogenic bacteria strains were the following (in mm): Pseudomonas syringae pv. syringae UCM B-1027T – 19±3, P. fluorescens IMV 8573 – 22±3, Pectobacterium carotovorum UCM B-1075T – 17±2. Activity against Xantomonas campestris pv. campestris UCM B-1049, Clavibacter michiganensis subsp. michiganensis IMV 102, Agrobacterium tumefaciens UCM B-1000 was not detected. Minimum inhibitory concentrations (MIC) in the range from 8 to 64 μg/mL for P. carotovorum UCM B-1075T, Erwinia aroidea IMV 1058, Proteus vulgaris UCM B-905 and P. fluorescens IMV 8573 are hardly comparable with the discovered against staphylococci. TLC analysis of its broth extract revealed five separate compounds with different values of retention factors: Rf1=0.42; Rf2=0.38; Rf3=0.31; Rf4=0.28; Rf5=0.25. The main component of extract was batumin, other four substances were present in minor quantities. All found substances had similar absorption maxima with the minimum differences between isomeric forms: descarbamoyl batumin-enol (Mr=505, λ=226 nm), descarbamoyl batumin-keto (Mr=505, λ=231 nm), batumin (Mr=548, λ=231 nm), batumin-enol (Mr=548, λ=228 nm) and 17-hydroxy-batumin (Mr=550, λ=229 nm). The largest inhibition zone (P. carotovorum UCM B-1075T) was on the third compound placement which represents of batumin, tiny inhibition zones were found around keto and enol form of descarbamoyl batumin. Observation of live bacterial cells in light microscope confirmed a serious disruption of motility in all these bacteria by batumin in the concentration far lower than the MIC for these organisms. Proteus actively moved in the control, but in presence of 10 μg/mL of batumin was almost no growth. The biofilm formation by P. batumici UCM B-321 was stimulated by supplementing batumin into the medium. The stimulation effect by batumin on the biofilm formation was equally strong when the compound was applied in the concentrations of 1 and 10 μg/mL. Batumin was not an attractant of the producer strain. However, in one of our experiments batumin has shown the properties of positive effector (attractant) for B. subtilis UCM B-7023 strain. Conclusion. The discovered features allow to consider the antibiotic batumin synthesized by P. batumici UCM B-321 as the essential tool for survival and competition of the producer strain in a natural habitat.

Keywords: Pseudomonas batumici, batumin, biofilm formation, antimicrobial activity, phytopathogenic bacteria.

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