Trans-aconitic acid assimilation system as a widespread bacterial mechanism for environmental adaptation

Abstract

The ability of bacteria to use natural carbon sources greatly affects their growth and survival in the environment. Bacteria have evolved versatile abilities to use environmental carbon sources, but their diversity and assimilation pathways remain largely unexplored. Trans-aconitic acid (TAA), a geometric isomer of cis-aconitic acid involved in the tricarboxylic acid cycle, has long been considered a natural carbon source metabolizable by bacteria. However, its catabolism and ecological role in linking bacterial interactions with the environment remain unclear. Here, we identify a regulatory system in Bacillus velezensis FZB42 that is capable of sensing and catabolizing TAA. The system consists of a tar operon, an adjacent positive regulatory gene tarR, and a shared promoter. After receiving the TAA signal, the TarR protein interacts directly with the promoter, initiating the expression of the membrane transporter TarB and aconitate isomerase TarA encoded by the operon, which function in importing the TAA and isomerizing it into the central intermediate cis-aconitic acid. Subsequent soil colonization experiments reveal that TAA assimilating ability can give its coding bacteria a growth and competitive advantage. Bioinformatics analyses coupled with bacterial isolation experiments further show that the assimilation system of TAA is widely distributed in the bacterial domain, and its assimilating bacteria are also extensively distributed in nature, indicating an important role of TAA metabolism in bacterial carbon acquisition. This work emphasizes the importance of metabolic adaptation to environmental carbon sources for bacterial survival and may provide inspiration for engineering microbes with enhanced environmental competitiveness.

Keywords: Bacillus velezensis; trans-aconitic acid; trans-aconitic acid importer TarB; aconitate isomerase TarA; bacterial inducible operon tar; carbon source assimilation; metabolic adaptation; microbial survival.

Graphical Abstract

Figure 1

Genetic validation of the B. velezensis FZB42 TAA assimilation gene cluster. (A) Chemical structures of TAA and CAA geometric isomers. The configurational difference of the 1-carboxyl and the 6-carboxyl groups in TAA and CAA molecules on the opposite side and the same side of the double bond are highlighted. (B) Genetic organization of the tar genes in the FZB42 genome. Distances are shown to scale. Genes flanking the tarR and tar operon are: RBAM_RS16110, LacI family DNA-binding transcriptional regulator coding gene; RBAM_RS16115, ATP-dependent Clp endopeptidase proteolytic subunit ClpP coding gene; RBAM_RS16135, TIGR00730 family Rossman fold protein coding gene; and RBAM_RS16140, MazG-like family protein coding gene. Two pairs of green inverted arrows indicate the amplified regions in the tar operon analysis, and the sizes of amplified products F1R1 and F2R2 are given. (C) Structure diagram of the tar promoter. (D) TAA utilization phenotypes of FZB42, tar gene mutants, and their corresponding complementary strains on ACO medium plates.

Figure 2

AI activity of the TarA protein. (A) HPLC analysis of AI activity of TarA in an in vitro catalytic system. (B, C) LC-Q-TOF-MS verification of CAA formation in a reverse system and TAA formation in a forward system, respectively. The values next to the chromatographic peaks indicate the retention times. The m/z 173.0088 ion is the mass of the [M-H]⁻ ion of aconitic acid. The signals at m/z 129.0191 and 85.0298 are the decarboxylation products of one and two carboxyl groups from the [M-H]⁻ ion, respectively.

Figure 3

Characterization of TarB as a TAA membrane importer. (A) Fluorescence micrograph of TarB-GFP fusion protein in the FZB42-TarB-GFP strain, and (B) GFP protein in the FZB42-GFP strain. Scale bar, 2 μm. (C) Western-blot confirmed the presence of TarB in the cell membrane fraction. M: membrane fraction, C: cytoplasmic fraction. (D) MST revealed the binding effect and constant between TarB protein and TAA substrate. (E) LC-Q-TOF-MS analysis of the presence of TAA in the intracellular fractions of FZB42 and ΔtarB strains. Both cells were examined at the 10 h time point of the growth phases in Fig. 3F. The ion at m/z 173.0088, represents the mass of aconitic acid in [M-H]⁻ mode and was used to extract CAA and TAA signals from the total ion chromatogram (TIC). The retention times of the extracted CAA and TAA signals are provided. (F) Growth curves and (G) TAA residual levels in the supernatants of FZB42 and ΔtarB strains cultured in modified ACO liquid medium.

Figure 4

Regulatory properties of TarR. (A) Secondary structure analysis of TarR using the PROSITE tool of the Expasy database. The helix-turn-helix (HTH) motif and DNA-binding site at the N-terminus, and substrate binding region at the C-terminus are indicated. (B) Relative expression of tarA and tarB genes in FZB42, ΔtarR, and CtarR strains, and (C) the tarR gene in FZB42 and CtarR strains, with (+) and without (−) TAA induction. The transcription level of tar genes in FZB42 without TAA induction was defined as 1. The 23S rRNA gene was used as a reference. (D, E) In vitro EMSA assays to determine the specific binding of TarR to the P_tar DNA region. (F) DNase I foot-printing assay of the protected region of the tarA promoter by TarR (red boxes). (G) EMSA detection of the interaction between TarR and mutant P_tar DNA, P_tar(#1/2/3)M, in which the three palindromic sequences of “TTATAA” were changed to “CCGCGG”.

Figure 5

TAA directly binds to TarR protein. (A) MST analysis of the interaction between TarR and TAA, with citric acid (B) as a control. (C) EMSA tests the effect of TAA molecules on enhancing TarR-P_tar interaction, with citric acid (D) as a control.

Figure 6

Phylogenetic distribution of TAA assimilation genes in bacteria. The numbers above each column are the total number of targets in the phylum, including tarA-type (containing only tarA homolog), tarAB-type (containing only “tarA + tarB” homolog), and tarRAB-type (containing “tarR + tarA + tarB” homolog) species. The dot plots show the relative abundances of TAA assimilation functions within each phylum.

Figure 7

Individual and competitive colonization of the TAA assimilator FZB42 and its mutant ΔtarA disabled in TAA assimilation in sterile soils with and without TAA addition. (A) and (B), individual colonization in sterile soils without and with TAA addition, respectively. (C) and (D) competitive colonization in sterile soils without and with TAA addition, respectively. (E) Ratio of the number of FZB42 cells to the number of ΔtarA cells counted in (D).

Figure 8

Proposed models for how TAA assimilation confers survival advantage on bacteria in natural environment. TAA metabolites can be secreted by bacteria (such as B. thuringiensis and Pseudomonas spp.) or by the roots of plants (such as maize, grass, and sugar cane) as a carbon source. Returning the plant tissues to the field can also increase TAA content. When TAA is present, both bacterial consumers (e.g. B. velezensis) and non-consumers that do not encode AI enzymes will use the most common carbon sources (e.g. glucose and citric acid), but only the former is able to use TAA. Therefore, in the same living space, TAA assimilation can give consumer bacteria more nutrients, thereby achieving more vigorous growth and larger population size than non-consumer bacreria, and eventually winning the survival competition. Specifically, the molecular mechanism by which bacteria absorbing environmental TAA as a carbon source is as follows. Step 1. Perception. The detection of TAA molecules may be achieved by leaky expression of TarB protein, as evidenced by the detectable tarB transcripts by RT-PCR in the absence of TAA (see Fig. S11). Step 2. Activation. In the cytoplasm, the imported TAA acts as a signal to bind directly to the TarR protein, a positive specific regulator of TAA assimilation function. TarR then directly binds to P_tar to significantly activate tar expression and the production of TarA and TarB proteins. Step 3. Transport. TarB importers robustly transport available environmental TAA into the cell. Step 4. Isomerization. TarA enzymes serve as an AI to convert TAA into CAA, incorporating the carbon source into the central metabolism of the TCA cycle, and promoting the production of more building materials and energy.