Spermidine promotes Bacillus subtilis biofilm formation by activating expression of the matrix regulator slrR

Abstract

Ubiquitous polyamine spermidine is not required for normal planktonic growth of Bacillus subtilis but is essential for robust biofilm formation. However, the structural features of spermidine required for B. subtilis biofilm formation are unknown and so are the molecular mechanisms of spermidine-stimulated biofilm development. We report here that in a spermidine-deficient B. subtilis mutant, the structural analogue norspermidine, but not homospermidine, restored biofilm formation. Intracellular biosynthesis of another spermidine analogue, aminopropylcadaverine, from exogenously supplied homoagmatine also restored biofilm formation. The differential ability of C-methylated spermidine analogues to functionally replace spermidine in biofilm formation indicated that the aminopropyl moiety of spermidine is more sensitive to C-methylation, which it is essential for biofilm formation, but that the length and symmetry of the molecule is not critical. Transcriptomic analysis of a spermidine-depleted B. subtilis speD mutant uncovered a nitrogen-, methionine-, and S-adenosylmethionine-sufficiency response, resulting in repression of gene expression related to purine catabolism, methionine and S-adenosylmethionine biosynthesis and methionine salvage, and signs of altered membrane status. Consistent with the spermidine requirement in biofilm formation, single-cell analysis of this mutant indicated reduced expression of the operons for production of the exopolysaccharide and TasA protein biofilm matrix components and SinR antagonist slrR Deletion of sinR or ectopic expression of slrR in the spermidine-deficient ΔspeD background restored biofilm formation, indicating that spermidine is required for expression of the biofilm regulator slrR Our results indicate that spermidine functions in biofilm development by activating transcription of the biofilm matrix exopolysaccharide and TasA operons through the regulator slrR.

Keywords: Bacillus subtilis; agmatine; aminopropyl; bacteria; biofilm; exopolysaccharide; polyamine; slrR; spermidine; transcriptomics.

Figure 1.

Polyamine structures and biosynthesis in B. subtilis. A, relevant polyamine structures. The first methylene carbon of spermidine is by convention designated as the 1 position and is on the aminopropyl side of the molecule. B, pathway for spermidine biosynthesis in B. subtilis. C, synthetic polyamine analogues. In the agmatine analogues, the number of methylene carbons in the potential diamine chain are indicated in parentheses. Aminopropyl groups are shown in blue, aminobutyl groups in red, and aminopentyl groups in tan.

Figure 2.

The effect of exogenous polyamines on biofilm formation of polyamine auxotrophic strains derived from B. subtilis NCIB3610. Wild-type undomesticated strain NCIB3610, gene deletion of arginine decarboxylase (strain ΔspeA), and gene deletion of S-adenosylmethionine decarboxylase (strain ΔspeD). A, effect of exogenous natural polyamines (100 μm) on biofilm formation. Spd, spermidine; Nspd, norspermidine; Hspd, homospermidine; Nspm, norspermine; Spm, spermine; Put, putrescine; Cad, cadaverine. B, effect of exogenous synthetic methylated spermidine analogues and agmatine structural analogues (100 μm) on biofilm formation. 1-MeSpd, 1-methylspermidine; 2-MeSpd, 2-methylspermidine; 3-MeSpd, 3-methylspermidine; 8-MeSpd, 8-methylspermidine; Agm, agmatine, Noragm, noragmatine; Homoagm, homoagmatine. Colony biofilms were developed on solid MSgg agar containing 100 μm natural or synthetic polyamines. Wild-type NCIB3610 colonies are ∼1 cm in diameter and the different sizes of other colonies are proportional to the wild-type.

Figure 3.

Proposed biosynthesis of agmatine analogue-derived polyamines in B. subtilis and results of precursor feeding in the ΔspeA strain. A, proposed pathway for conversion of agmatine structural analogues to diamines and triamines. B, exogenous (500 μm) agmatine but not putrescine is converted to spermidine in ΔspeA as detected by HPLC. Fl, fluorescent label; Put, putrescine; Spd, spermidine. C, growth of ΔspeA with exogenous (100 μm) homoagmatine (Homoagm) but not noragmatine (Noragm) results in the production of a new peak (shown by vertical) arrow in the HPLC detection of polyamines. Fl, fluorescent label; Spd, spermidine. D, LC-MS analysis of ΔspeA with or without growth in 100 μm homoagmatine (Homoagm). Extracted ion chromatograms (472/473) for the detection of protonated tribenzoylated aminopropylcadaverine (m/z 473.3). The mass spectrum (inset in lower panel) of the peak at 3.527 min, found only after growth with homoagmatine, reveals masses for protonated tribenzoylated aminopropylcadaverine (m/z 473.3) and its sodium adducted form (m/z 494.3).

Figure 4.

Expression of both the tapA-sipW-tasA and eps(A-O) operons is reduced in spermidine auxotrophic colony biofilms. Flow cytometry analysis of single cells harvested from complex colonies grown at either 37 °C for 17 h (A and B) or 30 °C for 40 h (C and D). A, C, and D, expression from the tapA promoter; B, expression from the epsA promoter. Strains analyzed were: NCIB3610, NRS2242 (3610, sacA::P_epsA-gfp), NRS3972 (speA::spc, sacA::P_epsA-gfp), NRS2394 (3610, sacA::P_tapA-gfp], NRS4100 (ΔspeD, sacA::P_tapA-gfp] and NRS3974 (speA::spc, sacA::P_tapA-gfp). C and D, the exogenous addition of spermidine (Sp), norspermidine (Nsp) or homospermidine (Hsp) to the growth medium is indicated. The NCIB3610 non-fluorescent control sample shown in C and D is the same, as these experiments were run simultaneously.

Figure 5.

Restoration of slrR expression in the speD mutant results in restoration of colony biofilm complexity and expression of both the eps(A-O) and tapA-sipW-tasA operons. A, disruption of sinR results in a hyper-complex complex colony in both an otherwise wild-type strain and in the ΔspeD mutant. Biofilms were grown at 30 °C for 48 h prior to imaging. Strains included: NCIB3610, NRS5332 (sinR::kan), NRS4005 (ΔspeD), and NRS5330 (ΔspeD, sinR::kan). B, restoration of slrR expression in ΔspeD mutant restores colony biofilm complexity. Expression of slrR was induced by the addition of IPTG to the MSgg agar, and complex colonies were grown at 30 °C for 48 h prior to imaging. Strains included: NCIB3610, NRS4005 (ΔspeD) and NRS5331 (ΔspeD, amyE::P_hyspank-slrR-lacI). C-F, heterologous expression of slrR in the wild-type (C, D) or speD mutant (E, F) results in unimodal expression of both the tapA-sipW-tasA (C, E) and eps(A-O) (D, F) operons. Flow cytometry analysis of single cells harvested from complex colonies grown at 37 °C for 17 h, either in the absence of IPTG induction or with induction of slrR expression with the addition of 10 or 25 μm IPTG. Strains analyzed were: NCIB3610, NRS2394 (3610, sacA::P_tapA-gfp), NRS5338 (3610, sacA::P_tapA-gfp amyE::P_hyspank-slrR-lacI), NRS5337 (3610, sacA::P_epsA-gfp amyE::P_hyspank-slrR-lacI), NRS5053 (ΔspeD, amyE::P_hyspank-slrR-lacI, sacA::P_tapA-gfp), NRS2242 (3610, sacA::P_epsA-gfp), and NRS5334 (ΔspeD, amyE::P_hyspank-slrR-lacI, sacA::P_epsA-gfp). In C–F the NCIB3610 sample is the same in all four graphs as experiments were run simultaneously. In C and E the same wild-type sample is shown in both for ease of comparison with other samples. In D and F the same wild-type sample is shown in both to facilitate comparison.