Traditionally, T. rubrum phenotypes were mostly detected using carbon source assimilation tests, during which the types of tested substances are limited, the operation is complicated, and only one substance can be tested at once. PMs overcome this defect by detecting the color response during the respiratory metabolism of living cells, enabling the collection of large amounts of data on the microbial utilization of various nutrients. In the present study, we analyzed the metabolic differences between the laser treated and non-treated (control) T. rubrum groups. First, compared with laser-irradiated T. rubrum, the control group efficiently utilized saccharides (e.g., arabinose, D-fructose, sucrose) and organic acids (e.g., glucuronic acid, D-malic acid, and aminobutyric acid) as carbon sources as well as some amino acids (e.g., alanine, arginine, serine) as nitrogen sources for growth. Many sulfur and phosphorus source substrates were also utilized, but their utilization degree was lower than that of the carbon and nitrogen source substrates. Second, compared with control T. rubrum, the number of substrates utilized by T. rubrum after laser irradiation were significantly reduced, including saccharides, organic acids, and a variety of amino acids, indicating that the process of energy generation may be damaged by laser [15]. Third, the substrate utilization degree in the laser treated group was higher than that in the control group for several substances, such as carbon source substrate D-glucosamine, nitrogen source substrates L-aspartic acid and L-glutamine, phosphorus source substrate D-glucosamine-6-phosphate, and sulfur source substrate D-methionine. This could be related to a self-protection mechanism or the fact that laser irradiation promotes T. rubrum apoptosis. Further studies could help us better understand whether this is self-protection or cell death, and the associated molecular pathway.

Results from the carbon source test showed that the types of metabolized substrates were similar between the two groups, but the number of metabolized substrates was different. The laser treated group only used 30% of the substrates used by the control group (Figs. 1 and 2). Saccharides are the most abundant carbon source and may play a key role in the survival of T. rubrum. The tricarboxylic acid cycle (TCA) is not only a common metabolic pathway in aerobic organisms but also the final metabolic pathway of three major nutrients (saccharides, lipids, and amino acids). After the laser irradiation of T. rubrum, a variety of metabolites involved in the TCA were inhibited, including alpha-ketoglutarate, D-malic acid, succinic acid, L-asparagine, L-proline, L-threonine, and putrescine. Therefore, we speculate that after laser irradiation, some substrates cannot be utilized by T. rubrum due to the damages of genes that modulate the TCA, reducing the metabolic capacity of T. rubrum. In addition, laser irradiation reduced the metabolic capacity of T. rubrum for some substrates involved in glycolysis, such as D-glucuronic acid and maltitol. On this basis, it is speculated that laser irradiation may destroy multiple key genes involved in glycolysis or the TCA of T. rubrum, thus affecting the metabolism of corresponding substrates. Although the TCA is the main glucose metabolism pathway, it is not the only one. When the TCA is inhibited, the pentose phosphate pathway can be used to metabolize carbohydrates [16]. In this study, T. rubrum irradiated by laser could still utilize some carbon source substrates in the pentose phosphate pathway, including D-arabinose, L-arabinose, D-ribose, and D-xylose.

D-glucosamine exists widely in the chitin and glycoproteins of bacterial cell walls and the chitin of fungal cell walls in the form of N-acetylglucosamine [17]. The transport of glucosamine to cells through glucose transporters [18] will directly increase the flux through the hexosamine biosynthesis pathway, enhancing O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation [19]. The O-GlcNAc protein is an endogenous protective mechanism triggered by stress and is considered an “emergency receptor” [20]. Increases in O-GlcNAc levels have been shown to contribute to higher heat resistance in cells [20]. In our study, we showed that the utilization of D-glucosamine is increased after laser irradiation. We suspect that laser irradiation of T. rubrum triggers this self-protection mechanism, resulting in a higher degree of D-glucosamine utilization.

Microbial growth and product synthesis require nitrogen sources, which are mainly used for the biosynthesis of amino acids, proteins, nucleic acids, and nitrogen metabolites. Research on the sporulation of T. rubrum under different nitrogen sources has shown that T. rubrum can grow normally without a carbon source when nitrogen sources are available [21], suggesting that nitrogen substrates are vital to T. rubrum. In our PM3 microplate with nitrogen sources, the control group effectively utilized 20 nitrogen-containing substances, mainly amino acids involved in glucose and nucleic acid metabolism and a few dipeptides. However, only 12 nitrogen substrates were utilized by T. rubrum after laser irradiation, and the degree of metabolism of most substrates was significantly lower than that of the control group.

Amino acids are precursors for purine and pyrimidine biosynthesis. Amino acid metabolism participates in the TCA and is the central point of glucose, lipid, and amino acid metabolism. After laser irradiation, T. rubrum lost the ability to utilize alanine, arginine, asparaginate, isoleucine, proline and ornithine, and its degree of utilization of glutamate, glycine, and serine decreased, indicating that amino acid metabolism was inhibited. Moreover, laser irradiation increased the utilization of L-aspartic acid and L-glutamine by T. rubrum, which may be due to the key roles of glutamine and aspartic acid in cell growth and proliferation. Glutamine and aspartic acid, as intermediate metabolites of glucose metabolism, can participate in the TCA and provide energy for cells [22]. They also provide nitrogen sources for nucleotides, proteins, and other biological macromolecules [23]. The efficient utilization of glutamine and aspartic acid can also affect DNA repair and replication [24] to maintain the survival of bacteria.

Phosphorus absorption and utilization play important roles in biological processes, such as heredity, energy metabolism, cell membrane integrity, and intracellular signal transduction. Organisms have formed a complex phosphate system to regulate phosphorus metabolism [25]. Our analysis showed that without laser irradiation, T. rubrum effectively utilized phosphate, trimetaphosphate, hypophosphate, adenosine-3’-monophosphate, adenosine-2’,5’-cyclic monophosphate, D-2-phosphoglyceric acid, D-glucose-6-phosphoric acid, 2-deoxy-D-glucose-6-phosphoric acid, 6-phosphoric acid-gluconic acid, creatine phosphate, and choline phosphate, which are involved in gluconeogenesis, phospholipid metabolism, nucleotide metabolism, energy transport, and signal transduction. After laser irradiation, the ability of T. rubrum to utilize these phosphorous substances was lost or significantly reduced.

Sulfur-containing amino acids include methionine, cysteine, and cystine. Methionine is one of the most easily oxidized amino acids in organisms and the activity center of proteins. Methionine in proteins can function normally only if the correct structure is maintained. However, under oxidative stress, methionine is very likely to be oxidized to methionine sulfoxide [26]. The increase in methionine utilization capability in the laser treated group may be related to increased ROS levels after laser irradiation [6, 27]. ROS can oxidize methionine to methionine sulfoxide, which may affect a variety of biological functions [28]. Methionine sulfoxide reductase is widely distributed in pathogens and can reduce methionine sulfoxide to methionine. Under normal conditions, methionine sulfoxide reductase can reverse the above-mentioned oxidation reaction, protecting against oxidative stress [29]. However, after laser irradiation, the capability of T. rubrum to utilize methionine sulfoxide decreased, and this oxidation could not be prevented or reversed (Fig. 7).

Methionine and glutathione synthesis pathways. Hollow black arrows indicate the direction of reaction; Solid arrow (↑) indicates utilization in the laser treated group is increased, while (↓) indicates utilization in the laser treated group is decreased compared with the control

Glutathione is a low-molecular-mass polypeptide composed of glycine, cysteine, and glutamic acid. Glutathione can remove superoxide ions and other free radicals to prevent damage to the cell [30]. Studies have shown that the laser irradiation of T. rubrum leads to an increase in ROS [6, 31]. We expect T. rubrum in the laser treated group to utilize more glutathione to resist the damage caused by superoxidation. However, our results revealed that the degree of glutathione utilization in the laser group was lower than that in the control group, which may be because glutathione is affected by multiple metabolic enzymes. The laser irradiation of T. rubrum could lead to glutathione synthesis dysfunction, limiting the ability to maintain the reduction under oxidative stress and reducing the glutathione utilization capacity of the laser group.