Sfp-type PPTase inactivation promotes bacterial biofilm formation and ability to enhance wheat drought tolerance

Abstract

Paenibacillus polymyxa is a common soil bacterium with broad range of practical applications. An important group of secondary metabolites in P. polymyxa are non-ribosomal peptide and polyketide derived metabolites (NRPs/PKs). Modular non-ribosomal peptide synthetases catalyze main steps in the biosynthesis of the complex secondary metabolites. Here we report on the inactivation of an A26 Sfp-type 4'-phosphopantetheinyl transferase (Sfp-type PPTase). The inactivation of the gene resulted in loss of NRPs/PKs production. In contrast to the former Bacillus spp. model the mutant strain compared to wild type showed greatly enhanced biofilm formation ability. A26Δsfp biofilm promotion is directly mediated by NRPs/PKs, as exogenous addition of the wild type metabolite extracts restores its biofilm formation level. Wheat inoculation with bacteria that had lost their Sfp-type PPTase gene resulted in two times higher plant survival and about three times increased biomass under severe drought stress compared to wild type. Challenges with P. polymyxa genetic manipulation are discussed.

Keywords: Evolution Canyon; Paenibacillus polymyxa; Sfp-type PPTase; natural isolate genetic manipulation; plant drought tolerance; rhizobacterial biofilm.

Figure 1

Schematic drawing of the shuttle plasmid pGEM7Z-sfp and the inactivation strategy.

Figure 2

Phenotypic and chemical analysis of P. polymyxa A26 Sfp-type PPTase mutant (A26Δsfp). (A) Inhibitory effect of wild type A26 (a), A26Δsfp (b) and complemented strain A26Δsfp/pHPS9-sfp (c) against F. graminearum. Note that the zone of antagonism observed with wild type has disappeared with mutant and is fully restored with complemented strain. (B) Antimicrobial activities of fusaricidin, polymyxin and MALDI-TOF MS analysis of culture extracts of wild type A26 (a1 and a2, respectively; blue line) and A26Δsfp (b1 and b2; green line). Note that the fusaricidins (molecular weights 883, 897, 911, 931, 947, and 961 Da) and polymyxin (molecular weight 1094 Da) produced in wild type are eradicated in A26Δsfp culture extracts. (C) Antimicrobial activities of fusaricidin, polymyxin and MALDI-TOF MS analysis of complemented strain A26Δsfp/pHPS9-sfp culture extracts (c1 and c2; blue line) is compared to mutant A26Δsfp (b1 and b2; green line). Note that synthesis of fusaricidins and polymyxin is fully restored by A26Δsfp strain complemented with plasmid pHPS9-sfp.

Figure 3

Biofilm and root hair formation analysis of P. polymyxa Sfp-type PPTase mutants. (A) In vitro biofilm formation of A26 (a), A26Δsfp (b), A26Δsfp/pHPS9-sfp (c), E681 (d), E681Δsfp (e), compared to B. subtilis 3610 (f), and 3610Δsfp (g). Colony phenotypes of the strains are shown. Colonies were grown on PDA agar for 4 days at 30°C. The scale bar represents 2 mm. (B) Scanning electron microscopic images of A26 (a), A26Δsfp (b), A26Δsfp/pHPS9-sfp (c) inoculated wheat roots. Significantly more biofilm compared to A26 is formed on the roots inoculated with A26Δsfp; complementation of the strain with pHPS9-sfp restores the wild type biofilm formation level. The scale bar represents 3. (C) Light microscopic images of biofilm and root hair formation on wheat roots inoculated A26 (a), A26Δsfp (b), and A26Δsfp/pHPS9-sfp (c). Note that compared to A26 significantly more root hair and biofilm are formed on wheat roots inoculated with A26Δsfp. Complementation of A26Δsfp restores the wild type of root hair and biofilm formation levels.

Figure 4

P. polymyxa A26 and its Sfp-type PPTase mutant (A26Δsfp) wheat drought tolerance enhancement analysis. Effect of A26 and A26Δsfp inoculation on wheat dry mass (A) and survival (B).

Figure 5

Direct effect of A26 Sfp-type PPTase mediated metabolites on wheat root. Scanning electron microscopic images, dry mass, root bacterial counts and survival of A26 (A), A26Δsfp metabolite extract treated roots compared to (B), A26 (C), and A26Δsfp bacterial culture (D) treated roots.

Figure 6

Direct effect of polymyxin B and polymyxin E on wheat root. Three concentrations of the antibiotics (0.3, 2.5 and 7.5 μg/ml) were used in the study. Effect of polymyxin B (A) and polymyxin E (B) on shoot and root dry weight under well watered regime and drought stress. Note that due to germination impairment by polymyxin E only lowest concentration of polymyxin E treated plant dry weights of shoots and roots are shown.