The Rcs System Contributes to the Motility Defects of the Twin-Arginine Translocation System Mutant of Extraintestinal Pathogenic Escherichia coli

Abstract

Flagellum-mediated bacterial motility is important for bacteria to take up nutrients, adapt to environmental changes, and establish infection. The twin-arginine translocation system (Tat) is an important protein export system, playing a critical role in bacterial physiology and pathogenesis. It has been observed for a long time that the Tat system is critical for bacterial motility. However, the underlying mechanism remains unrevealed. In this study, a comparative transcriptomics analysis was performed with extraintestinal pathogenic Escherichia coli (ExPEC), which identified a considerable number of genes differentially expressed when the Tat system was disrupted. Among them, a large proportion of flagellar biosynthesis genes showed downregulation, indicating that transcription regulation plays an important role in mediating the motility defects. We further identified three Tat substrate proteins, MdoD, AmiA, and AmiC, that were responsible for the nonmotile phenotype. The Rcs system was deleted in the Δtat, the ΔmdoD, and the ΔamiAΔamiC strains, which restored the motility of ΔmdoD and partially restored the motility of Δtat and ΔamiAΔamiC. The flagella were also observed in all of the ΔtatΔrcsDB, ΔmdoDΔrcsDB, and ΔamiAΔamiCΔrcsDB strains, but not in the Δtat, ΔmdoD, and ΔamiAΔamiC strains, by using transmission electron microscopy. Quantitative reverse transcription-PCR data revealed that the regulons of the Rcs system displayed differential expression in the tat mutant, indicating that the Rcs signaling was activated. Our results suggest that the Rcs system plays an important role in mediating the motility defects of the tat mutant of ExPEC. IMPORTANCE The Tat system is an important protein export system critical for bacterial physiology and pathogenesis. It has been observed for a long time that the Tat system is critical for bacterial motility. However, the underlying mechanism remains unrevealed. In this study, we combine transcriptomics analysis and bacterial genetics, which reveal that transcription regulation plays an important role in mediating the motility defects of the tat mutant of extraintestinal pathogenic Escherichia coli. The Tat substrate proteins responsible for the motility defects are identified. We further show that the Rcs system contributes to the motility suppression. We for the first time reveal the link between the Tat system and bacterial motility, which is important for understanding the physiological functions of the Tat system.

Keywords: Rcs system; extraintestinal pathogenic Escherichia coli; flagella; motility; transcription regulation; twin-arginine translocation system.

FIG 1

Differentially expressed genes (Δtat versus WT). (A) KEGG pathway enrichment of the differentially expressed genes. The differentially expressed genes were analyzed by using the BlastKOALA tool (https://www.kegg.jp/blastkoala/) to assign K numbers which were then used for pathway enrichment by using KEGG Mapper (https://www.genome.jp/kegg/mapper/search.html). The numbers inside the vertical bars are the total numbers of genes belonging to each level 2 KEGG pathway. (B) COG enrichment of the differentially expressed genes. COG enrichment was performed with the EggNOG v5.0 database (http://eggnog5.embl.de/#/app/home).

FIG 2

Disruption of the Tat system interferes with the expression of the flagellar biosynthesis genes. (A) In the swimming assay, 5 μL of cells of each indicated strain at the mid-log phase were spotted onto the swimming plate (tryptone, 10 g/L; yeast, 5 g/L; NaCl, 10 g/L; agar, 0.2%), which was dried at room temperature for 5 min followed by incubation at 37°C for 7 h. The CΔtat strain is the Δtat strain harboring a plasmid expressing tatABC in trans. The assay was performed in triplicate, and the data shown are representative. (B) RT-qPCR analysis. Total RNA was extracted from cells of each indicated strain grown to the mid-log phase. Genomic DNA was removed, and cDNA was synthesized, which was used as the template for qPCR with a QuantStudio 6 Flex fluorescence quantitative PCR instrument. The expression level of the tested gene was calculated using the 2^−ΔΔCT method and normalized to the housekeeping gene gapA. Data are displayed as the geometric mean ± standard error of the mean (SEM). Statistical significance between the two groups was analyzed using Student’s t test. ***, P  < 0.001. (C) Analysis of flagellin production. Cells of each indicated strain were grown to the mid-log phase in LB at 37°C with shaking. The culture was centrifuged, and the same amount of culture supernatant was collected and concentrated by ultracentrifugation. The same number of bacterial cells was harvested, resuspended in PBS, and lysed by sonication, and these were the whole-cell samples. Samples were normalized and subjected to SDS-PAGE followed by Western blotting using anti-FliC antibody (cat. no. ab93713; Abcam).

FIG 3

Identification of Tat substrate proteins responsible for loss of motility. (A) For the swimming assay with the Tat substrate-related mutants, 5 μL of cells of each indicated strain grown to the mid-log phase was spotted onto the swimming plate (tryptone, 10 g/L; yeast, 5 g/L; NaCl, 10 g/L; agar, 0.2%) and was dried at room temperature for 5 min followed by incubation at 37°C for 7 h. The assay was performed in triplicate, and the data shown are representative. (B) Swimming assay with ΔamiAΔamiC, ΔmdoD, and the strains in trans expressing the corresponding proteins. The CΔmdoD and CΔmdoD^RR-KK strains are the ΔmdoD strain encompassing a plasmid encoding the wild type and the mutated MdoD in which the twin-arginine in the signal peptide is replaced with a twin-lysine. CΔamiAΔamiC is the ΔamiAΔamiC strain in trans expressing AmiA and AmiC. (C) and (D) RT-qPCR analysis. Total RNA was extracted from cells of each indicated strain grown to the mid-log phase. Genomic DNA was removed, and cDNA was synthesized and was used as the template for qPCR with a QuantStudio 6 Flex fluorescence quantitative PCR instrument. The expression level of the tested gene was calculated using the 2^−ΔΔCT method and normalized to the housekeeping gene gapA. Data are displayed as the geometric mean ± SEM. Statistical significance between the two groups was analyzed using Student’s t test. ***, P < 0.001.

FIG 4

Disruption of the Rcs system restored motility. First, 5 μL of cells of each indicated strain grown the mid-log phase was spotted onto the swimming plate (tryptone, 10 g/L; yeast, 5 g/L; NaCl, 10 g/L; agar, 0.2%) and was dried at room temperature for 5 min followed by incubation at 37°C for 7 h. The assay was performed in 5 replicates for each strain. The representative image is presented on the left. The swimming diameter is measured and shown as the mean ± standard deviation on the right. ***, P < 0.001.

FIG 5

Transmission electron microscopy analysis of flagella. Cells of each indicated strain were streaked on an LB agar plate at 37°C for 12 h. A single colony was picked and resuspended in 200 μL of ultrapure water, which was left to stand for 2 h. Then, 10 μL of the bacterial suspension was placed on the grid followed by fixing with 2% phosphotungstic acid staining solution and was imaged using a transmission electron microscope (H-7650; Hitachi, Japan). The scale bar is 1 μm.

FIG 6

RT-qPCR analysis of the Rcs regulons. Total RNA was extracted from cells of each indicated strain grown to the mid-log phase. Genomic DNA was removed, and cDNA was synthesized and was used as the template for qPCR with a QuantStudio 6 Flex fluorescence quantitative PCR instrument. The expression level of the tested gene was calculated using the 2^−ΔΔCT method and normalized to the housekeeping gene gapA. Data are displayed as the geometric mean ± SEM. Statistical significance between the two groups was analyzed using Student’s t test. ***, P < 0.001.