Type I fimbriae subunit fimA enhances Escherichia coli biofilm formation but affects L-threonine carbon distribution

Abstract

The biofilm (BF) provides favorable growth conditions to cells, which has been exploited in the field of industrial biotechnology. Based on our previous research works on type I fimbriae for the biosynthesis of L-threonine (LT) in Escherichia coli, in this study, a fimA-overexpressing strain was engineered, which improved BF formation under industrial fermentation conditions. The morphological observation and characterization of BF formation were conducted to verify the function of the subunit FimA. However, it was not suitable for repeated-batch immobilized fermentation as the LT titer was not elevated significantly. The underlying molecular mechanisms of BF formation and the LT carbon flux were explored by transcriptomic analysis. The results showed that fimA regulated E. coli BF formation but affected LT carbon distribution. This study will stimulate thoughts about how the fimbriae gene regulated biofilms and amino acid excretion and will bring some consideration and provide a reference for the development of BF-based biomanufacturing processes in E. coli.

Keywords: Escherichia coli; L-threonine carbon distribution; biofilm; fimA gene; transcriptomic analysis.

FIGURE 1

Semi-quantitative analysis of BF formation in the three strains. (A) Crystal violet staining in the two media. (B) Fluorescence microscopy of the BFs stained by DAPI.

FIGURE 2

TM3000 electron microscopy of cell adhesion and BF formation at various magnification levels. First column: W1688; second column: W1688-fimA*; third column: W1688-△fimA. Cell adhesion and BF formation were noticeable in E. coli W1688-fimA*. Cells were encased in BFs by extracellular secretions with the form of aggregation with each other. BF formation was decreased and presented as a sparse distribution in E. coli W1688-ΔfimA.

FIGURE 3

SEM (A) and TEM (B) images of BF formation and cell adhesion. First row: SEM; second row: TEM. First column: W1688; second column: W1688-fimA*; third column: W1688-△fimA. (C) RT-qPCR validation of the fimA expression. (D) RT-qPCR validation of flagellar gene expression in WT and the fimA* strain. Mean ± SD (n = 3). Two-tailed t-test. ***p < 0.001, **p < 0.01, and *p < 0.05. The results of SEM for the observation of BF formation were similar to crystal violet staining of the BF. Type I fimbriae were tiny on the cell surface (Figure 3B1, the red arrow). The absence of fimbriae with smooth appearance was observed in W1688-△fimA (Figure 3B3). An increase was observed in the extracellular surface fimbriae in W1688-fimA* (Figure 3B2, the red arrow), and the accumulation of the flagellum assembly with the filamentous substance attached to bacteria was observed in W1688-fimA*, which intertwined cells with each other (Figure 3B2, the blue arrow).

FIGURE 4

Levels of LT and glucose produced and consumed by the three strains during batch fermentation, respectively.

FIGURE 5

Quantitative analysis of crystal violet staining for BFs and dry cell weight in the three different strains.

FIGURE 6

Transcriptomic analysis of genes responsible for the BF formation pathway in the three strains. Blue and red denote downregulated and upregulated genes, respectively.

FIGURE 7

Transcriptomic profiles of LT biosynthetic pathways in three different strains. (A) Transcriptomic analysis of the three different strains. Blue and red denote downregulated and upregulated genes, respectively. From the top to the bottom panels: genes in the biosynthesis of LT, L-aspartate, L-cysteine, L-arginine, and acetate biosynthesis and the TCA cycle, respectively; (B) LT biosynthetic pathways in E. coli. Blue and red arrows indicate downregulated and upregulated genes, respectively. (C) RT-qPCR validation of the genes associated with LT biosynthesis and transportation. Mean ± SD (n = 3). Two-tailed t-test. ***p < 0.001, **p < 0.01, and *p < 0.05.