Recent taxonomic studies have revealed that B. subtilis is heterogeneous and should be considered as a group of closely related species . In addition, B. subtilis and B. amyloliquefaciens are phenotypically similar and can be easily confused. Based on phenotypic and biochemical characterisations, strain GA1, initially isolated from strawberry cultures, was first assigned to B. subtilis (unpublished data, ). However, substantial molecular evidence suggested that the strain was more related to B. amyloliquefaciens than to B. subtilis. Thus, to accurately characterise the strain taxonomically, recA and recN genes, encoding DNA repair and recombination proteins, were sequenced and used to construct a phylogenetic tree. These two genes were selected because they had previously been shown to be effective in resolving closely related taxa . The obtained phylogenetic tree clearly demonstrates that strain GA1 should be assigned to B. amyloliquefaciens rather than B. subtilis. The higher level of identity obtained for the 358 protein-coding sequence detected in strain GA1 to that of B. amyloliquefaciens FZB42 is in accordance with this result.
Plant-associated bacteria are known to play a key role in plant health by stimulating their growth and protecting them from phytopathogens, with this related to the production of a vast array of biologically active NRPS and PKS secondary metabolites. These metabolites have the same mode of synthesis, the so-called multicarrier thiotemplate mechanism, in which small monomer units, aminoacids and aryl acid in NRPS and acyl-CoAs in PKS are loaded, activated and condensed by mega-enzymes organised in iterative functional units . The aim of this work was to better genetically characterise B. amyloliquefaciens GA1, with particular emphasis on gene clusters encoding these mega-enzymes. As they are encoded by large operons of 55 to 80 kb for PKS and 18 to 42 kb for NRPS, randomly sequencing 10% of the genome yielded enough data to point out the presence of these gene clusters in strain GA1.
Among NRPS antibiotics, Bacillus amyloliquefaciens GA1 was found to produce surfactin, iturin A, fengycin A and fengycin B. These are cyclic lipopeptides (CLP) composed of seven (surfactin and iturin A) or 10 α-amino acids (fengycins) linked to a β-amino (iturins) or β-hydroxy (surfactins and fengycins) fatty acid which may vary from C-13 to C-16 for surfactins, from C-14 to C-17 for iturins and from C-14 to C-18 for fengycins. CLPs have well-recognized potential biotechnology and biopharmaceutical applications due notably to their surface-active properties [24, 25]. These surfactants may also play different roles in the development and survival of Bacillus strains in their natural habitat: increasing bioavailability of hydrophobic water-insoluble substrates, heavy metal binding, bacterial pathogenesis, quorum sensing, motility, biofilm formation etc. [26, 27]. Other works have highlighted additional traits that are also very important for the fitness of Bacillus in the rhizosphere and for its efficacy as biocontrol agent .
The ability of B. subtilis to efficiently colonise surfaces of plant roots is a prerequisite for phytoprotection. This process relies on surface motility and efficient biofilm formation of the Bacillus cell populations that evolve and behave as structured and coordinated microcolonies adhering to root and on soil particle surfaces . By modifying cell surface properties, surfactin and iturin were reported to positively influence cell spreading, swarming and biofilm formation [30–33] and thus may globally favour plant root colonisation. Furthermore, iturins and fengycins display strong antifugal activity and are inhibitory for the growth of a wide range of plant pathogens. Surfactins are not fungitoxic in themselves but retain some synergistic effect on the antifungal activity of iturin A . In the context of biocontrol, the involvement of CLPs in direct antagonism of phytopathogens is thus obvious and was demonstrated by testing the pure compounds in planta or by correlating the biocontrol activity and use of non-producing or overproducing derivatives [5, 35, 36].
The role of fengycins produced by strain GA1 was demonstrated by the very effective disease control provided by treatment of fruits with CLP-enriched extracts and by in situ detection of fengycins in inhibitory amounts . Another recently established role for lipopeptides from beneficial Bacillus isolates is the stimulation of the plant immune system . Surfactins and, to a lesser extent, fengycins can induce a priming state in host plant which allows an accelerated activation of defense responses upon pathogen or insect attack, leading to an enhanced resistance to the attacker encountered . Surfactins can be considered as a novel class of microbial-associated molecular patterns that can be specifically perceived by plant cells as signals to activate defense mechanisms . The use of single strains evolving diverse mechanisms to reduce disease incidence is thus of prime interest. Bacillus isolates such as strain GA1 that co-produce the three CLP families should display such a multi-faceted biocontrol activity. That said, in strain GA1 the itu operon directing the synthesis of iturin A in B. subtilis RB14  was surprisingly found inserted at exactly the same position as the expected bacylomycin D gene cluster from B. amyloliquefaciens FZB42 . This suggests that an inter-species horizontal transfer of genes could have occurred between B. subtilis and B. amyloliquefaciens.
Besides lipopepides, a functional dhb gene cluster was shown in strain GA1 to direct the synthesis of the catecholic siderophore bacillibactin, a cyclic trimeric lactone of the 2,3-dihydroxybenzoyl-Gly-Thr monomer unit. Siderophores are high affinity ferric iron chelators that enhance the microbial acquisition of this element in environments where its bioavailability is extremely low, e.g. in soils. Thus, the presence of siderophore-producing microorganisms in the rhizosphere contributes to plant health by complexing iron and making it less available to phytophathogens that are generally not able to produce comparable Fe-transport systems .
In addition to peptides, polyketides are the other dominant family of secondary metabolites having revelant bioactivities. Similarly to B. amyloliquefaciens FZB42, three functional gene clusters directing the synthesis of difficidin, macrolactin and bacillaene were identified in strain GA1. Difficidin is an unsaturated 22-membered macrocylic polyene lactone phosphate ester  with broad spectrum antibacterial activity . It inhibits protein biosynthesis  and was recently shown promising in its suppressive action against Erwinia amylovara, a devasting plant pathogen causing necrotrophic fire blight disease of apple, pear and other rosaceous plants . By contrast, macrolactin and bacillaene have not yet been demonstrated to be directly related to biocontrol, although they are both antimicrobial agents that could be potentially useful in human medicine. Macrolactin, which consists of a 24-membered ring lactone, had the ability to inhibit murine melanoma cancer cells as well as mammalian herpes simplex viruses. It was also shown effective in protecting lymphoblast cells from HIV . Similarly to difficidin, bacillaene is an inhibitor of prokaryotic protein synthesis constituted by an open-chain enamine acid with an extended polyene system. This compound displays antimicrobial activity toward human pathogens such as Serratia marcescens, Klebsiella pneumoniae and Staphylococcus aureus .
Bacilysin is a dipeptide composed of an L-alanine and the unusual amino acid L-anticapsin and represents one of the simplest peptide antibiotics known with antifungal and antibacterial activities . L-anticapsin, released after uptake in susceptible cells, is an inhibitor of the glucose amine-6-phosphate synthetase, an enzyme essential in cell wall biosynthesis . Due to its antibacterial activity, bacilysin is effective as a biocontrol agent, notably by inhibiting the growth of E. amylovora, the causative agent of fire blight disease . It is also effective on the Absidia ssp., which is responsible for cutaneous and invasive zygomycosis in immunocompromised patients . Besides bacilysin, some strains of B. subtilis also co-produced chlorotetaine, a chlorinated derivative of bacilysin with similar antibacterial activity [20, 49]. Although no direct evidences are available, some experimental data suggest that the two compounds could share the same biosynthetic pathway . Here, mass spectrometry analysis demonstrated that B. amyloliquefaciens GA1 synthetises only chlorotetaine as dipeptide antibiotic. While this behaviour is not clearly understood, this is to our knowledge the first B. amyloliquefaciens strain reported to produce chlorotetaine and the first strain to produce chlorotetaine and not bacilysin.