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 6RRERH11R 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 83DI85SLWIEK91G 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.


| INTRODUC TI ON
Salmonella belongs to the member of the family Enterobacteriaceae and is an important zoonotic agent of public health concern. Nontyphoid Salmonella accounted for over 59,000 deaths and they were with the highest rank for disability adjusted life years among the foodborne disease hazards in 2010 (Kirk et al., 2015). Salmonella enterica contains more than 2,579 serovars while S. Typhimurium is one of the major serovars of Salmonella that account for infections in human and animals (CDC, 2011). Adhesion to the host cell is the first step to establish infection for many bacteria including S. Typhimurium. Adhesion molecules on the surface of bacteria or conjugated on the shaft of a structure called fimbria have been implicated in engaging such an adherent event. In addition, signal transduction in bacteria can also be mediated by adhesion to the host cell (Moorthy, Keklak, & Klein, 2016).
Fimbriae are hair-like appendages present on the outer membrane of many bacteria. S. Typhimurium has been documented to possess 13 different fimbrial gene clusters that may have the potential to produce fimbriae (McClelland et al., 2001). Among these fimbriae types, type 1 fimbriae with the binding specificity to mannosylated residue is frequently found and is also referred to as the common fimbriae (Duguid, Anderson, & Campbell, 1966). The fim gene cluster in the genome of S. Typhimurium is responsible for the phenotypic expression of type 1 fimbriae. The fim gene cluster consists of genes for structure, biosynthesis, and those for regulation. FimA, FimI, FimF, and FimH are fimbrial structural subunits that incorporate each other to form a fimbrial shaft. Fimbrial subunits are assembled and anchored in the outer membrane of bacteria by chaperone protein FimC and usher protein FimD, respectively (Clegg & Swenson, 1994). The genes fimZ, fimY, fimW, stm0551, and fimU have been shown to regulate type 1 fimbrial production through a complicated circuit involving both the transcriptional and translational levels and protein-protein interaction as well Tinker, Hancox, & Clegg, 2001;Yeh, Hancox, & Clegg, 1995). FimY is a positive regulator for type 1 fimbrial expression in S. Typhimurium. The C-terminal part of FimY possesses a helixturn-helix motif and FimY was shown to bind to the promoter region of fimZ (Wang, Hsu, Huang, Lin, & Yeh, 2014); however, some aspects of its mechanism in terms of fimbrial regulation remains to be elucidated. For example, the members of the PilZ family regulator, such as YcgR of Escherichia coli and MrkH of Klebsiella pneumoniae (Ryjenkov, Simm, Romling, & Gomelsky, 2006;Wilksch et al., 2011), often contain two conserved domains RRxxxR and DxSxxG (x designates any amino acid), which involve in binding cyclic di-GMP (c-di-GMP), a second messenger that controls many physiological processes of bacteria (Jenal, Reinders, & Lori, 2017). According to the protein sequence alignment, the N-terminal of FimY also possesses some amino acids that are present in these motifs (Figure 1). FimY harbors a 6 RRERH 11 R and a 83 DI 85 SLWIEK 91 G motif, with the second motif having extra three amino acids as compared with the DxSxxG motif; yet, its role as a c-di-GMP-binding protein has not been reported. Interestingly, a gene, stm0551, located just upstream of fimY was proven to encode a phosphodiesterase with an EAL domain whose function is to degrade c-di-GMP (Wang, Hsu, Huang, & Yeh, 2012). Nevertheless, there is no protein resembling a diguanylate cyclase to synthesize c-di-GMP within the fim gene cluster.
The present study reported here attempts to uncover the potential roles of 6 RRERH 11 R and 83 DI 85 SLWIEK 91 G of FimY and investigate how it may affect type 1 fimbrial expression in S. Typhimurium.
We found that the 7th arginine at the 6 RRERH 11 R motif of FimY may affect the production of type 1 fimbriae by activating fimZ and subsequently resulting in the fimbrial major subunit gene fimA expression.

| Construction of a S. Typhimurium fimY-deleted strain
The S. Typhimurium fimY-deleted strain was constructed by a one-step gene inactivation method (Datsenko & Wanner, 2000).
Kanamycin resistance gene from pKD13 tagged with a flanking sequence of the fimY gene was generated by a polymerase chain reaction (PCR) technique using fimY-P1 and fimY-P4 and transformed by electroporation into S. Typhimurium LB5010 that has previously been transformed with pKD46, the plasmid that encodes a λ red recombinase. The primers and PCR conditions are listed in Table 2.
All transformants were grown on LB agar supplemented with kanamycin. The constructed mutants were verified by PCR with primers located in the flanking sequence of the fimY gene.

| Construction of the recombinant plasmids possessing the fimY PilZ-like domain mutant alleles
The mutant alleles of fimY were generated by site-directed mutagenesis using an overlapping extension PCR with LB5010 strain genomic DNA (Ho, Hunt, Horton, Pullen, & Pease, 1989). The primers are listed in Table 2. Briefly, fimYSalI-F and R7A-R were used to amplify the first DNA fragment, while the second DNA fragment was amplified using fimYBamHI-R and R7A-F primers. Ligation of these

| Electron microscopy
Salmonella strains were prepared for electron microscopy using the negative staining procedure. Briefly, the bacterial culture was resuspended in ddH 2 O and mixed with an equal volume of sodium phosphotungstate (2%). A drop of the suspension was placed on a glow-discharged carbon-coated copper grid, and surplus fluid was removed with filter papers. The grids were air-dried and observed with a JEOL JEM-1400 transmission electron microscope (JEOL Ltd, Tokyo, Japan) operated at 60 or 80 kV.

| Detection of type 1 fimbriae by microplate yeast agglutination test
To analyze the expression ability of type 1 fimbriae of Salmonella strains, a microplate agglutination test (MAT) using yeast cells was performed in this study. All the Salmonella strains were cultured in 10 ml LB broth at 37°C for 48 hr statically, followed by centrifugation at 3,000 rpm for 10 min. MAT was performed as follows: First, the bacterial suspensions were adjusted to OD 600 1 ± 0.05, fol-

| Quantitative RT-PCR analysis
Total RNA was extracted and purified from bacterial strains cul-

| The electron microscopy
All Salmonella strains were observed for the phenotypic expression

| Detection of type 1 fimbriae by microplate yeast agglutination test
Microplate yeast agglutination test was used to quantify the type 1 fimbriae expression in all Salmonella strains. Figure 4 demonstrated the base two logarithms of a geometric average titer for each sample. The ΔfimY and ΔfimY (pACYC184) strains did not produce type 1 fimbriae and no titer was detected. The titer of the ΔfimY (fimY) was 4.64, which was higher than that of the wild type (2.76). The titer of other ΔfimY transformants were as follows: R11A and DSG were the same as ΔfimY (fimY), R7/11A and MUT were nearly 4, and the lowest was R7A (3.17). Compared with the ΔfimY (fimY), whose titer was considered as a baseline, the decrease in the titer of R7A was over 50%, while R7/11A and MUT were close to 36%.

| Quantitative RT-PCR analysis
The

| Secondary structure analysis
If FimY serves as a PilZ domain-like protein in S. Typhimurium, the secondary structure prediction program would reveal similarities between the configurations of FimY and other well-known PilZ domain proteins, such as MrkH of K. pneumoniae, DgrA of Caulobacter crescentus, and VCA0042 of Vibrio cholerae (Heidelberg et al., 2000;Ryjenkov et al., 2006;Wilksch et al., 2011). Figure 6 shows that the secondary structure pattern of FimY did not match that of the other well-documented PilZ domain proteins-the RRxxxR at the N-terminal followed by 6-7 β-strands forming a β-barrel and a C-terminal α-helix (Amikam & Galperin, 2006). However, the N-terminal of FimY only contained an α-helix.
Typhimurium only stm0551 had been described to involve in cdi-GMP metabolism, whose product is a phosphodiesterase to degrade c-di-GMP (Wang et al., 2012). No PilZ domain protein had been reported previously. In this study, we found that the amino acid of FimY does possess a RRxxxR motif and another DxSxxxxxG sequence motif; however, the function of these motifs has not been explored. Therefore, it is interesting to investigate the function of these motifs in terms of type 1 fimbrial production.
Since FimY is a positive regulator for type 1 fimbrial produc-  (Chan et al., 2004;Christen et al., 2006). In addition, the positive charged head groups of arginine are required for transient binding to the phosphate groups of c-di-GMP (Shoemaker, Portman, & Wolynes, 2000). The R7A and R7/11A strains decreased the production of fimbriae and  Nevertheless, the arginine at position 7 of the 6 RRxxx 11 R motif has a crucial role in activating fimZ and consequently fimA, resulting in an expression of type 1 fimbriae in S. typhimurium.

ACK N OWLED G M ENTS
This work was supported by Ministry of Science and Technology, Taiwan, under the contract number MOST 107-2313-B-002-051.
The authors would like to thank Ms. Shu-Ting Kuo from the Animal Health Research Institute, Council of Agriculture, Taiwan for assistance with electron microscopy.

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no conflict of interest.