3.1. Taxonomy of Bacillus strain
To clarify the taxonomic position of the isolated Bacillus strain, 16S rRNA gene was sequenced. According to the screening performed in the GenBank database, the closest sequence to the obtained sequence was 16S rRNA of B. pumilus NBRC 12092 strain. The 16S rRNA gene sequence of B. pumilus GY was most closely related to the reference strain with 100% identity. Phylogenetic tree showing links with other members of the genus Bacillus and with other B. pumilus strains is presented in Fig. 1. The isolated strain was designated as B. pumilus GY.
Figure 1. Phylogenetic tree reflecting the taxonomic position of the most closely related Bacillus pumilus NBRC 12092 strain to the studied B. pumilus GY strain. The optimal tree with the sum of branch length = 0.21147444 is shown. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.
3.2. TNT transformation
The ability of B. pumilus GY to grow on a synthetic medium in the presence of various concentrations of TNT (20–200 mg/L) was assessed. Bacterial growth was observed at all TNT concentrations, decreasing to the minimum at 200 mg/L (Fig. 2A). Despite the growth suppression of B. pumilus GY, it was able to eliminates 20–50 mg/ml TNT and reduce higher concentrations by this amount (Fig. 2B). In medium with an initial TNT concentration of 20 mg/L, the xenobiotic disappeared after 4 h of cultivation. Afterwards, the strain began to grow rapidly. The TNT concentration of 50 mg/L was eliminated after 24 h of cultivation. Other TNT concentrations were transformed at 24 hours of growth by 40, 24, and 18% from an initial TNT concentration of 100, 150, and 200 mg/L, respectively.
Identification of transformation products of high TNT concentrations (100–200 mg/L) at 24 h of cultivation revealed 4-amino-2,6-dinitrotoluene (4-ADNT) (Fig. 3A), at the same time, there were no nitrite ions in the cultivation medium. This suggests that the transformation of xenobiotic follows the pathway of nitro group reduction with the formation of monoamine derivatives of TNT. The first step in nitro group reduction can be achieved through one-electron transfer (solid line) or two-electron transfer (dashed line) (Fig. 3B, according to Esteve-Nuñez et al. 2001).
The first mechanism produces a nitro anion radical that could react with oxygen to form a superoxide radical and the original nitroaromatic compound through a futile cycle (dotted line). If the mechanism occurs via the transfer of two electrons, the nitroso derivative formed is the first putative intermediate; following two consecutive electron transfers, a hydroxylamine and an aromatic amine are produced. The superoxide radical is capable of further dismutation, which leads to the formation of hydrogen peroxide H2O2 (Ziganshin et al. 2015; Naumenko et al. 2017; Naumenko et al. 2013; Khilyas et al. 2013).
3.4. Proteome profiling
To determine the up- and down-regulation of proteins under the influence of TNT, densitometric analysis was carried out using the PDQuest software, during which the images of acrylamide gels containing proteins of the control and experimental groups were compared. All identified proteins correspond to the reference ones from reference strain Bacillus pumilus NBRC 12092. Under the action of 200 mg/L TNT, we visually observed the downregulation of 46 proteins; in addition to these proteins, 24 upregulated proteins were detected in the presence of TNT (Supplementary, Fig. 1). According to the databases, out of 24 upregulated proteins 23 were identified (Table 1), and out of 46 downregulated ones, 41 were identified (Table 2).
Among the proteins upregulated by TNT, flavin mononucleotide (FMN)-dependent azoreductase was identified, which has nitro reductase activity, and nitrate reductase which is also able to reduce TNT nitro groups to amino derivatives. Most likely, these proteins took part in the formation of 4-ADNT which we detected at 24 h of cultivation using HPLC (Fig. 3A).
One of the enzymes that promote the removal of ROS is catalase, which has been found among TNT-upregulated proteins. NAD-dependent malate dehydrogenase involved in the regulation of catalase activity was also reliably identified. This enzyme converts malate to oxaloacetate to form NADH, which has an inducing effect on catalase. Among TNT-upregulated proteins, both components of the antioxidant system thioredoxin/thioredoxin reductase (Trx/TrxR) were found.
Considering that nucleic acids are targets for ROS, it was expected to detect induction of repair enzymes under the action of TNT. Indeed, an increased production of the mismatch repair system enzyme as well as of methyltransferase was detected.
In response to stress conditions, the elimination of oxidized RNA by degradosomes containing polynucleotide phosphorylase, enolase, RNA helicase and RNase E is a well-known mechanism. The enolase has been identified among the TNT-upregulated proteins. Bacterial Clp proteases are involved in the degradation of proteins that are aggregated and incorrectly assembled during folding, as well as of proteins damaged by oxidative stress, so it's quite clear that the induction of Clp protease expression together with chaperonin which carries out the assembly of both newly synthesized and denatured proteins during various stress conditions.
Among the proteins downregulated by TNT were many enzymes participating in basic microbial metabolism including glycolysis, pentose phosphate pathway, pyruvate metabolism and citrate cycle indicating the general decrease in metabolic activity of bacterium. For example, NADH: quinone oxidoreductase involved in the first step of the electron transport chain was inhibited. Additionally, TNT alters the metabolism of amino acids, inhibiting expression of alanine dehydrogenase and ornithine aminotransferase. Decreased levels of L3 protein of 50S ribosomal subunit and of S1 protein of 30S subunit were identified. Apparently, TNT-induced downregulation of ribosomal proteins may indicate decreased biosynthesis of all proteins.
Using STRING software, we estimated the percentage of proteins involved in various metabolic processes of the cell in control (without TNT) and experimental (200 mg/L TNT) groups (Fig. 5). When proteins are grouped according to their biological functions, it can be seen that control cells perform a wider range of functions compared to TNT-exposed bacterial cells, that is manifested in 11 functional groups (Fig. 5B) compared to 7 ones (Fig. 5A). Among the proteins of the experimental group, the predominant enzymes are those responsible for maintaining the redox state of the cell, the metabolism of the nitrogen-containing compound, and the degradation of RNA indicating a response to TNT-induced stress.
We also constructed a scheme of protein-protein interactions in experimental (Supplementary, Fig. 2A) and control group (Supplementary, Fig. 2B), which shows the changes in interteractome of B. pumilus GY with and without TNT.
Consequently, in the process of TNT transformation by B. pumilus GY, upregulated proteins are responsible for the reduction pathway of xenobiotic transformation, removal of oxidative stress and DNA repair, as well as RNA and proteins degradation. TNT inhibited the production of ribosomal proteins and proteins involved in in basic microbial metabolism.