Transposon Insertion in the purL Gene Induces Biofilm Depletion in Escherichia coli ATCC 25922

Abstract

Current Escherichia coli antibiofilm treatments comprise a combination of antibiotics commonly used against planktonic cells, leading to treatment failure. A better understanding of the genes involved in biofilm formation could facilitate the development of efficient and specific new antibiofilm treatments. A total of 2578 E. coli mutants were generated by transposon insertion, of which 536 were analysed in this study. After sequencing, Tn263 mutant, classified as low biofilm-former (LF) compared to the wild-type (wt) strain (ATCC 25922), showed an interruption in the purL gene, involved in the de novo purine biosynthesis pathway. To elucidate the role of purL in biofilm formation, a knockout was generated showing reduced production of curli fibres, leading to an impaired biofilm formation. These conditions were restored by complementation of the strain or addition of exogenous inosine. Proteomic and transcriptional analyses were performed to characterise the differences caused by purL alterations. Thirteen proteins were altered compared to wt. The corresponding genes were analysed by qRT-PCR not only in the Tn263 and wt, but also in clinical strains with different biofilm activity. Overall, this study suggests that purL is essential for biofilm formation in E. coli and can be considered as a potential antibiofilm target.

Keywords: E. coli; biofilm; curli fibers; purL; transposon insertion.

Figure 1

Scatter plot and marginal histograms of the mutants. Upper bars (green) indicate the number of strains in the respective percentage of biofilm formation. Right bars (blue) indicate the number of stains in the respective absorbance values. Each spot represents a mutant where red colour indicates low biofilm formation rates and blue corresponds to high biofilm formation rates. LF: low biofilm former; F: biofilm former; HF: high biofilm former; A: absorbance.

Figure 2

Fitness assay in Luria Bertani (LB) broth. Green represents the wild type curve; Red represents Tn463. Grey represents the other 19 low biofilm-former (LF) transposon mutants, including Tn263. Growth rates were statistically evaluated via two-tailed Student t-test.

Figure 3

De novo purine biosynthesis in E. coli. Adapted from Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway maps.

Figure 4

Fitness assay. (a) Growth curves in LB broth (b) Growth curves in M63 broth. (c) Growth curves in M63 broth supplemented with inosine (50 µg/mL). Growth rates were statistically evaluated via two-tailed Student t-test.

Figure 5

Biofilm formation of ΔpurL::cat mutant, Tn263, and the complemented strain ΔpurL/purL+ using different concentrations of inosine. Each point in the curve represents mean values of A_580nm after 48 h of incubation; vertical bars correspond to standard deviations. The dashed line shows the absorbance value of the wild type strain, used as control. The results were analysed by One-way ANOVAs followed by post hoc Dunnett’s multiple comparisons tests.

Figure 6

Curli production. Dark red colony in yeast and casamino acid agar (YESCA-CR AGAR) (ATCC and ΔpurL/purL+) represents a curli producer, and light pink colonies (Tn263 and ΔpurL::cat) were associated with the defective phenotype. The defective phenotype of both mutants was restored in the YESCA-CR + Inosine agar.

Figure 7

Two-dimensional sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) images. (a) wt strain; (b) Tn263. The arrows mark significant differences (ANOVA p < 0.05) among the strains detected by Progenesis SameSpots 4.6.206. Further information about each spot identified can be found in the supporting information (Supplementary Table S5).

Figure 8

Analysis of the expression of selected genes in the wt and Tn263 mutants and in clinical isolates. (a) Heatmap showing the expression levels of biofilm-related genes in the wt and Tn263 mutant strains. Expression is presented as a logarithm of their 2^−ΔΔCt. Each square shows a biological replicate (n = 3 vs 3). (b) Scatter plot showing the expression of selected genes in biofilm producer and non-biofilm producer clinical isolates. Expression is normalised relative to the average expression of the biofilm producer isolates per each gene (n = X vs X). Each clinical isolate was performed in triplicate. T test: * p< 0.05, ** p < 0.01, *** p < 0.001.