Isocoumarins form a large, diverse class of biologically active metabolites with more than 200 metabolites
[79][133]. However, fewer are reported from
B. subtilis. It has been reported that
B. subtilis specifically produce isopropyl-8-hydroxy-3,4-dihydroisocoumarins with an active side chain or functionalized amino acid
[80][134]. Amicoumacin A-C (
Supplementary Material Figure S3E,F) and amicoumacin F are dihydroisocoumarins initially reported from
B. pumilus [81][82][83][135,136,137]. Later, they were reported from
B. subtilis and determined its strong antibacterial activity against the gastric pathogen
Helicobacter pylori [84][138]. Biosynthesis of these bioactive metabolites has been recently reported from
B. subtilis 1779 via a genome mining approach
[85][139]. The BGC encoding amicoumacin was predicted to be 47.4 kb in size and consists of 12 open reading frames. Further, the investigator expressed the biosynthetic gene cluster in a heterologous host and obtained amicoumacin A–C. Based on these findings, they predicted the biosynthetic pathway for amicoumacin to be synthesized via hybrid PKS-NRPS modular enzymes
[85][139]. The biosynthetic gene cluster consists of eight modules that synthesize two discrete pre-amicoumcin molecules. The unique dihydroisocoumarins core is likely to be synthesized via removing the AmiJ-M megasynthase complex to produce an oxygenated PK chain that reorganizes into a cyclic dihydroisocoumarin
[85][139]. Amicoumacin exhibit promising anti-inflammatory and antibacterial activity and has gained increased interest as a pro-drug candidate. Specifically, amicoumacin A is more attractive due to its potent anticancer activity and antibacterial activity against MRSA, with MIC less than 1 µg/mL
[82][83][136,137]. The antibacterial mechanism of amicoumacin was recently shown to be bind on the ribosome and inhibit protein biosynthesis
[86][87][140,141]. Amicoumacin A and C exhibit antibacterial activity, while amicoumacin B is non-antibacterial. Interestingly, the hydroxy group in amicoumacin A and C is responsible for their antibacterial activity as the phosphate ester-containing amicoumacin B lacks antibacterial activity
[88][142].
6. Volatile Metabolites
6.1. Volatile Inorganic Metabolites
The volatile inorganic metabolites are a by-product of the primary metabolites. They are usually nitrogen and sulfur-containing compounds such as ammonia (NH
3), hydrogen sulfide (H
2S), and hydrogen cyanide (HCN). Nitrogen-containing compounds are mainly emitted in the top sediment layer by denitrifying
B. subtilis strains. The
B. subtilis group also produces nitric oxide, which induces a systemic acquired resistance in plants against
Ralstonia solanacearum [89][157]. In an oxygen-deficient environment, bacteria emit various volatile inorganic metabolites such as hydrogen sulfide and hydrogen. These compounds act as a precursor for amino acid, antimicrobial metabolites synthesis, or act as electron acceptors.
B. subtilis produce hydrogen sulfide from sulfate hydrolysis or a by-product of L-cysteine and L-methionine catabolism
[90][91][158,159].
6.2. Volatile Organic Metabolites
6.2.1. Terpenes and Terpenoids
Terpenoids, also known as isoprenoids, are widely produced by all living organisms
[92][168]. The end product of the deoxy xylulose phosphate pathway, isopentenyl pyrophosphate (IPP), and their isomer dimethylallyl pyrophosphate (DMAPP) usually act as precursors for terpenoid biosynthesis
[90][158]. However, terpenoids may also be synthesized from isoprene
[93][169]. As previously shown, the isoprene produced by
B. subtilis is not synthesized via the deoxy-xylulose phosphate pathway. Isoprenoid plays a vital role in physiological functions such as membrane fluidity, electron transport, light-harvesting, and cell signaling
[94][170]. The involvement of terpenoids in cell signaling is particularly important, as it is associated with some mutualistic, antagonistic, and multitrophic interactions
[95][171]. Isoprenoids may potentially be used as a flavor, nutraceuticals, fragrance, and therapeutic agent in malaria and cancer treatment
[96][172].
6.2.2. Nitrogen-Containing Metabolites
Nitrogen-containing metabolites can be distinguished based on their degree of cyclization. To date, three groups of non-cyclic (amine, amide, and imines) and five groups of cyclic compounds (pyrazines, azole, pyridines, pyrimidines, and pyridazines) have been detected. Pyrazines are broadly represented among mVOCs and are categorized into two subclasses: higher alkylated and lower alkylated pyrazines
[90][158]. These metabolites are characterized by a strong odor, and most of the
B. subtilis strains isolated from rhizosphere and fermented products have been considered as pyrazine producers
[97][98][179,180]. The compound 2,5-Dimethyl pyrazine was isolated from
B. subtilis strain, exhibiting strong antifungal activity and inhibiting
P. chlamydospora [99][181]. Pyrazine produced by B. subtilis strains also inhibits the growth of bacteria such as
E. coli,
S. aureus, and
P. valgaris [100][182].
6.2.3. Sulfur-Containing Metabolites
Sulfur-containing volatile organic metabolites are synthesized from two primary sources, i.e. organic or inorganic
[90][158]. These metabolites often originate from the catabolism of amino acids, such as methionine, and sometimes from cysteine. However, the inorganic sulfate and sulfite may also act as a precursor for sulfur metabolites. The
B. subtilis group produces numerous antifungal and antinematicidal sulfur metabolites such as S-methyl butanethioate, S-methyl thioacetate, 2-methyl disulfide, and 3-methyl trisulfide. Among these, dimethyl disulfide also exhibits antibacterial activity
[101][102][103][104][185,186,187,188]. It is known that dimethyl sulfide disrupts bacterial cell communication by decreasing the sum of acyl-homoserine lactone
[105][189].
6.2.4. Benzenoids
Benzenoids are a diverse subclass often linked with sulfur or/and nitrogen. Most of the benzenoids produced by the
B. subtilis group displayed antifungal activity, while some inhibited the growth of bacteria and nematodes. Their antimicrobial mode of action is partially characterized. Nevertheless, the fungal and bacterial cell disruption is documented after exposure to bVOCs.
The
B. subtilis strain CF-3 was evaluated for their volatile organic metabolites potential. The investigator identified a plethora of volatile compounds based on solid headspace microextraction. Among them, benzothiazole exhibiting strong antimicrobial activity against fruit fungal pathogens
[106][190]. The volatile organic compounds produced by
B. amyloliqueficiens were evaluated for their effect on the growth and pathogenicity of tomato bacterial pathogen
R. solanacearum. The results showed that the strain emits several volatile organic compounds, including 1, 3 dimethoxy benzenes, inhibiting 62–85% growth of the tomato pathogen
[107][191].
6.2.5. Ketones
The biosynthesis of ketones is usually the result of fatty acid decarboxylation. Acetoin and its oxidative form butanedion are synthesized during the anaerobic fermentation of pyruvate. The two pyruvate molecules are condensed and converted to acetolactate by the acetolactate synthase enzyme. The acetolactate is further decarboxylated to form acetoin. Ketones are mainly known to inhibit the growth of plant pathogenic fungi. However, its antibacterial activity is yet not reported. Two ketones metabolites, i.e., 2-decanone, and 2-nonanone, displayed 100% growth inhibition of
F. oxysporum [108][193].
6.2.6. Hydrocarbon Metabolites
Hydrocarbon metabolites include alkane, alkene, and alkyne metabolites usually derived from fatty acid degradation via elongation decarboxylation or head-to-head condensation. Hydrocarbons are the most stable volatile organic metabolites and tend to remain in their original architecture over a long period of time. They may be used as a biomarker to estimate the age of primeval bacteria
[109][196]. The
B. subtilis group secretes various types of hydrocarbons such as nonane, tridecane, tetradecane, 2-methylpropane, and cyclohexane. Hydrocarbons like alkane, alkene, nonane, and decane are gaining particular interest due to their use as antimicrobial agents and fossil fuels. Nonane and 8-methyl heptadecane were isolated from
B. velezensis and exhibited antifungal activity against several fungal pathogens
[110][197]. Likewise, 1 3-butadiene inhibits the growth of phytopathogen and, additionally, negatively influences the chemotoxicity of the fungal pathogens
[111][198].
7. Miscellaneous Metabolites
There are few bioactive metabolites produced by
B. subtilis that do not fit into any class. For instance, bacilysocin is neither synthesized via NRPS nor PKS. Instead, it is a phospholipid antibiotic that accumulates within the
B. subtilis cell and presents a unique example of a modified phospholipid. Bacilysocin’s structure is composed of a central glycerol linked with glyceryl phosphate and an
anteiso-fatty acid tail. The putative biosynthetic pathway for the bacilysocin initiate is from the conversion of phosphatidic acid to phosphatidylglycerol and then lysophospholipase (YtpA) convert phosphatidylglycerol to bacilysocin. The antimicrobial activity of bacilysocin is limited to Gram-positive bacteria and a few fungal strains, including
S. aureus,
Candida pseudotropicalis and
S. cerevisiae. To date, the biological role, absolute biosynthetic pathway, mode of action, and the specific activity of bacilysocin is unclear.