Arginine within a specific motif near the N-terminal of FimY is critical for the maximal production of type 1 fimbriae in Salmonella enterica serovar Typhimurium

Abstract

An important Salmonella serovar for both human and animals Salmonella Typhimurium possesses 13 gene clusters that have the potential to produce fimbrial structure, among which the type 1 fimbriae with the binding specificity to mannose residue is the most commonly found type. Six structural genes and five regulatory genes comprise the fim gene cluster that is responsible for the production of type 1 fimbriae in S. Typhimurium. The fimY gene encodes a positive regulator for type 1 fimbrial expression since a deletion in fimY abolished the production of fimbriae. The N-terminal portion of FimY contains amino acid residues that exhibit some similarity as those found in the proteins possessing the PilZ domain, which is engaged in cyclic di-GMP binding. A fimY allele that had a change from arginine to alanine at position 7 (R7A) or 7 and 11 (R7/11A) generated by site-directed mutagenesis in a ⁶ RRERH¹¹ R motif near N-terminal, when cloned in pACYC184 and transformed into a fimY-deleted strain, decreased the expression of fimA and fimZ. The number of type 1 fimbriae in these two transformants was also less than those of the other transformants that contained different fimY alleles in pACYC184 when observed in electron microscopy. However, changing from arginine to alanine at position 11 (R11A) remained the same as the wild-type fimY allele. It is likely that the arginine at the 7th position of FimY is critical for its maximal activating activity upon fimZ. Another motif ⁸³ DI⁸⁵ SLWIEK⁹¹ G motif did not affect the function of FimY. Although FimY has the two aforementioned motifs, which contain some amino acids that are present within those of the PilZ domain proteins, secondary structure prediction analysis did not reveal that FimY has a conformation commonly observed in PilZ-like proteins. Therefore, FimY and PilZ domain proteins are not homologs. Further investigation for a detailed analysis of FimY is thus warranted.

Keywords: fimY; PilZ domain; Salmonella Typhimurium; type 1 fimbriae.

Figure 1

Conservation of PilZ‐containing domain proteins. Residues that show strict identity are written in white characters and highlighted in red. Similarity is indicated with black characters and highlighted in yellow. Residues that are crucial for c‐di‐GMP binding in the PilZ domain are marked with an asterisk. Protein names and microorganisms are as follows: FimY‐LT2, FimY of Salmonella Typhimurium LT2; MrkH‐KP, MrkH of Klebsiella pneumoniae AJ218; YcgR‐K12, YcgR of Escherichia coli K‐12

Figure 2

Observation of type 1 fimbriae by electron microscopy. (a) S. Typhimurium LB5010 wild‐type strain produced type 1 fimbriae on the outer membrane of the cell. (b) ΔfimY did not produce type 1 fimbriae. (c) There are no type 1 fimbriae present in the ΔfimY (pACYC184). (d) ΔfimY (fimY) resumed the ability to express type 1 fimbriae. Bacterial cells were negatively stained with 2% of phosphotungstic acid (60,000 x–80,000 x). Fimbriae are indicated by arrow

Figure 3

Observation of type 1 fimbrial expression in the Δ fimY strains that harbored plasmids expressing different fimY allels by electron microscopy. (a) R7A strain (b). R11A strain (c). R7/11A strain (d). DSG strain (e). MUT strain. All the strains produced type 1 fimbriae, whereas R7A and R7/11A strains produced comparatively less fimbriae than the others. Bacterial cells were negatively stained with 2% of phosphotungstic acid (60,000 x‐80,000 x)

Figure 4

Detection of type 1 fimbriae by microplate yeast agglutination test. The numbers indicate the geometric average of the titer end point (N = 3) of different Salmonella strains

Figure 5

Quantitative real‐time polymerase chain reaction analysis of fimA and fimZ of different S. Typhimurium strains. The expression of (a) fimZ and (b) fimA was calculated using the ^ΔΔCt method and designed as the fold change compared with the geometric mean expression level of the ΔfimY (fimY) strain in triplicate. A p value < 0.05 was considered to represent a significant difference. *p < 0.05; **p < 0.01; ***p < 0.001

Figure 6

Secondary structure prediction. Protein size of 100 amino acids beginning from ⁶RRERH¹¹R of FimY in Salmonella Typhimurium (accession No.: AAL19504), ¹⁰⁶RRRDP¹¹¹R of MrkH in Klebsiella pneumoniae (accession No.: AEO27488), ¹¹RRAHP¹⁶R of DgrA in Caulobacter crescentus (accession No.: AE005673.1), and ¹³⁴QLRKEP¹⁴⁰R of VCA0042 in Vibrio cholerae (accession No.: NP_232443) were analyzed using PHD to predict their secondary structures; red: β‐strand; pink: random coli; blue: α‐helix.