The role of nitric-oxide-synthase-derived nitric oxide in multicellular traits of Bacillus subtilis 3610: biofilm formation, swarming, and dispersal

Frank Schreiber,Martin Beutler,Dennis Enning,José González-Pastor,María Lamprecht-Grandio,Dirk De Beer,Olga Zafra

doi:10.1186/1471-2180-11-111

Abstract

BackgroundBacillus subtilis 3610 displays multicellular traits as it forms structurally complex biofilms and swarms on solid surfaces. In addition, B. subtilis encodes and expresses nitric oxide synthase (NOS), an enzyme that is known to enable NO-mediated intercellular signalling in multicellular eukaryotes. In this study, we tested the hypothesis that NOS-derived NO is involved in the coordination of multicellularity in B. subtilis 3610.ResultsWe show that B. subtilis 3610 produces intracellular NO via NOS activity by combining Confocal Laser Scanning Microscopy with the NO sensitive dye copper fluorescein (CuFL). We further investigated the influence of NOS-derived NO and exogenously supplied NO on the formation of biofilms, swarming motility and biofilm dispersal. These experiments showed that neither the suppression of NO formation with specific NOS inhibitors, NO scavengers or deletion of the nos gene, nor the exogenous addition of NO with NO donors affected (i) biofilm development, (ii) mature biofilm structure, and (iii) swarming motility in a qualitative and quantitative manner. In contrast, the nos knock-out and wild-type cells with inhibited NOS displayed strongly enhanced biofilm dispersal.ConclusionThe results suggest that biofilm formation and swarming motility in B. subtilis represent complex multicellular processes that do not employ NO signalling and are remarkably robust against interference of NO. Rather, the function of NOS-derived NO in B. subtilis might be specific for cytoprotection against oxidative stress as has been proposed earlier. The influence of NOS-derived NO on dispersal of B. subtilis from biofilms might be associated to its well-known function in coordinating the transition from oxic to anoxic conditions. Here, NOS-derived NO might be involved in fine-tuning the cellular decision-making between adaptation of the metabolism to (anoxic) conditions in the biofilm or dispersal from the biofilm.

Highlights

Bacillus subtilis 3610 displays multicellular traits as it forms structurally complex biofilms and swarms on solid surfaces
Gusarov and Nudler [6] showed that Nitric oxide (NO) synthase (NOS)-derived NO in B. subtilis provides instant cytoprotection against oxidative stress imposed by H2O2 with two different mechanisms
NO formation in B. subtilis 3610 We tested intracellular production of NO in B. subtilis 3610 grown in LB and in Minimal salt glycerol glutamate (MSgg) medium by staining cells with the NO sensitive dye CuFL

Summary

Introduction

Bacillus subtilis 3610 displays multicellular traits as it forms structurally complex biofilms and swarms on solid surfaces. B. subtilis encodes and expresses nitric oxide synthase (NOS), an enzyme that is known to enable NO-mediated intercellular signalling in multicellular eukaryotes. Nitric oxide (NO) is a signalling molecule in multicellular, eukaryotic organisms, where it coordinates the function and interactions between cells of the cardiovascular, neuro, and immune system [1]. These cells have the ability to synthesize NO with the enzyme NO synthase (NOS) using arginine and O2 as substrates [2]. The targets of NO signalling are mainly NO-mediated protein modifications, such as iron-nitrosylation and S-nitrosylation of active site cysteine thiols These modifications critically depend on the apparent NO concentration and the redox conditions. A completely different function of NOS-derived NO was described in Streptomyces turgidiscabies, where it is involved in the biosynthesis of a secondary metabolite (a dipeptide phytotoxin) by the site-specific nitration of a tryptophanyl moiety [8]

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