A z66-positive wild-type strain, S. Typhi GIFU10007, was used in this study. Mutants and plasmids used in this work are listed in Table 1. Primer sequences are described in Table 2. Bacteria were cultured in Luria–Bertani (LB) broth at 37°C.

The stp17 gene deletion mutant of S. Typhi was prepared through homologous recombination mediated by the suicide plasmid pGMB151, as previously described (Huang et al., 2004; Zhang et al., 2012a). The stp17 deletion mutant was confirmed by sequencing analysis and designated as Δstp17.

For complementary expression of stp17 in Δstp17, the CDS of stp17 was amplified with pfu DNA polymerase by PCR. The amplicon was inserted into the complementary vector pACYC184 to form the recombinant plasmid pACYC184-stp17, which was verified by sequencing analysis. Then, the strain Δstp17 was transformed by the recombinant plasmid pACYC184-stp17 and designated as the stp17 complementary strain Δstp17(pACYC184-stp17). As a control, the strain Δstp17 was also transformed with the empty vector pACYC184 and named Δstp17(pACYC184).

The stp17 ORF (a 642-bp DNA fragment) was cloned into the expression vector pBAD/gIII which can be induced by L-arabinose to generate the recombinant plasmid (pBAD-stp17). The recombinant plasmid pBAD-stp17 was confirmed by sequence analysis and subsequently transformed into the wild type strain S. Typhi GIFU10007 to generate the over-expression strain wt(pBAD-stp17). As a control, the strain S. Typhi GIFU10007 was also transformed with the empty vector pBAD/gIII and named wt(pBAD). Over-expression of stp17 in wt(pBAD-stp17) was induced by L-arabinose (0.1% wt/vol).

The entire stp17 ORF (a 642-bp DNA fragment) containing a Nco I site (5′-end) and a Xho I site (3′-end) was amplified by PCR. Then, the resulting PCR product was digested with Nco I and Xho I and cloned into plasmid pET28b which carries a N-terminal His-tag digested with the same enzymes. The resulting stp17 recombinant expression plasmid pET28b-stp17 was transformed into E. coli BL21(DE3) cells. The cell cultures were incubated at 37°C in LB medium until the OD₆₀₀ reached 0.6. To induce expression of the recombinant protein, IPTG was added at a final concentration of 0.03 mM. The culture was grown for 5 h at 37°C and harvested by centrifugation (4000 r/min, 10 min, 4°C). Bacteria were re-suspended in 20 ml PBS. After bacteria were lysed with an ultrasonic cell disruptor, bacterial lysate was purified using a Ni-NTA agarose column as directed by the manufacturer (QIAGEN). Recombinant protein STP17_His6 was eluted with elution buffer containing 250 mM imidazole. Purified STP17_His6 from the elution buffer was concentrated in PBS using Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-10 membrane, according to the manufacturer's protocol (Millipore Corporation, Bedford, MA, USA). Purified STP17_His6 was analyzed by SDS-PAGE.

To prepare the polyclonal antibody of STP17, purified STP17_His6 was mixed completely with an equal volume of Freund's complete adjuvant and 1 ml of mixture which contained 50 μg STP17_His6 was injected subcutaneously into each rabbit. After 1 week, purified STP17_His6 was mixed completely with an equal volume of Freund's un-complete adjuvant and the mixture was used to immunize the rabbits as above every 2 weeks. The rabbits were immunized for total five times. Finally, the antiserum was obtained from the carotid artery, and was purified through salting out extraction with ammonium sulfate to prepare the rabbit polyclonal antibody of STP17.

S. Typhi wild type and mutant Δstp17 strains were cultured overnight with shaking (250 rpm) at 37°C. Bacteria were normalized to OD₆₀₀ 0.6. Proteins were separated by 15% SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked with 5% dried skim milk. Rabbit anti-STP17 antiserum as the primary antibody was used at a dilution of 1:500 and incubated with the membrane for 2 h at room temperature. HRP-conjugated goat anti-rabbit-IgG antibody (Sigma-Aldrich, St. Louis, Missouri, USA) was used at a dilution of 1:10,000 as the secondary antibody. Horseradish peroxidase-antibody conjugates were detected by chemiluminesence.

The expression characteristics of STP17 protein in S. Typhi GIFU10007 under different growth phases were investigated by western blot. S. Typhi cells were collected when their OD₆₀₀ values were 0.2, 0.5, 0.8, 1.2, and 1.6, respectively. Western blotting experiments in triplicate were performed as previously described.

The plasmid curing method was referred to the reference (El-Mansi et al., 2000) with some following modifications. Sodium dodecyl sulfate (SDS) was added to LB media and the final concentration of SDS was adjusted to 1%. The wild-type strain S. Typhi GIFU10007 was inoculated into the above SDS containing LB and cultures were incubated overnight with shaking at 43°C. Subsequently, the cultures were inoculated into normal LB and incubated overnight with shaking at 43°C. The cultures were inoculated into SDS containing LB again and cultured overnight at 43°C with shaking. Then, the cultures were placed on to SS agar plates to select the possible pBSSB1-cured derivatives of S. Typhi through the amplification of the gene fljB^z66 located on pBSSB1 by PCR. Finally, the possible pBSSB1-cured derivatives of S. Typhi were verified by Southern-blot as previous described (Zhang et al., 2012b) and designated as ΔpBSSB1. Then, the strain ΔpBSSB1 was transformed by the recombinant plasmid pACYC184-stp17 and designated as the stp17 complementary ΔpBSSB1 strain ΔpBSSB1(pACYC184-stp17). As a control, the strain ΔpBSSB1 was also transformed with the empty vector pACYC184 and named ΔpBSSB1 (pACYC184).

Based on the amino acid sequence of protein STP17 which were retrieved from GenBank (accession no. AM419040.1; Baker et al., 2007a), its secondary structure and three-dimensional structure were predicted by I-TASSER online server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) as previous described (Cai et al., 2013). In addition, the conserved ATPase domain of STP17 was predicted, and the possible ATP binding site residues of STP17 was analyzed through I-TASSER online server as well. According to the predicted possible ATP binding site residues of STP17, the site-directed mutations were introduced into pACYC184-stp17 using standard PCR mutagenesis techniques, and mutations were confirmed by DNA sequencing. The mutants generated were the following: Δstp17:: pACYC184-stp17^G16A, Δstp17:: pACYC184-stp17^G17A, Δstp17:: pACYC184-stp17^G19A, Δstp17:: pACYC184-stp17^K20A, Δstp17:: pACYC184-stp17^N133A, Δstp17:: pACYC184-stp17^R134A, Δstp17:: pACYC184-stp17^K157A, Δstp17:: pACYC184-stp17^K158A.

Bacteria were grown in LB medium at 37°C. A single colony of bacteria from a LB agar plate was inoculated into 2 ml of LB broth and incubated at 37°C with shaking (250 rpm) overnight. Then, the culture was diluted 1/100 in fresh LB broth (containing L-arabinose (0.1% wt/vol) for the culturing of strains harboring pBAD). Cell growth was monitored at OD₆₀₀ every hour using a BioPhotometer (Eppendorf, Hamburg, Germany). The experiments were repeated at least three times.

For detecting the motility of bacteria, different strains were incubated until their OD₆₀₀ reached 0.4. From each culture, 2 μl was inoculated onto a 0.3% semisolid LB agar plate (containing L-arabinose 0.1% wt/vol) for the strains harboring pBAD). The plates were incubated at 37°C for 10 h and motility was assessed qualitatively by examining the diameter of circular swimming, which was formed by growing motile bacterial cells. Meanwhile, the flagella gene fljB^z66 and fliC expression of different strains were examined by qRT-PCR.

The potential ATPase activity of STP17 was determined by the ATP assay kit (Beyotime, China) according to the manufacturer's recommended protocol. Briefly, STP17_His6 protein was added to the ATP solution provided in the assay kit at a concentration of 10 μM and then incubated at 25°C for 10-, 30-, and 50 min, respectively. The ATP concentration of the above reaction mixtures was measured by a F-4500 fluorescent spectrophotometer (Hitachi, Japan). As a control, BSA protein which lacks ATPase activity was mixed with the ATP solution and analyzed on the spectrophotometer. Because ATPase can catalyze ATP into ADP and free phosphate, the reduction of ATP concentration in the reaction mixture indicates the presence of ATPase activity, which should be dependent on the added protein being studied (STP17_His6). These experiments were performed at least three times.

Data were analyzed using Prism 5 software. The Student's t-test was used to determine significant differences between results. Significance was defined as P < 0.05.

The gene stp17 is located on a linear plasmid named pBSSB1 in S. Typhi and is predicted to encode a putative nucleotide binding protein, STP17. In this study, expression of stp17 in E. coli BL21(DE3) was generated with the expression vector pET28b. Results showed that the purified recombinant protein STP17_His6 was successfully obtained (Figure 1A). To verify whether the stp17 gene is expressed and translated into STP17 protein in S. Typhi, western blotting was performed to identify STP17 levels in the S. Typhi wild type strain and the mutant strain Δstp17 (Figure 1B). The results showed that the gene stp17 can be translated into STP17 protein in S. Typhi.

We investigated the expression characteristics of STP17 in different growth phases by western blot analysis. Five OD₆₀₀ values (0.2, 0.5, 0.8, 1.2, and 1.6) corresponding to early log phase, mid-log phase, late log phase, early stationary phase and late stationary phase, respectively, were monitored. As shown in Figure 2, stp17 can be expressed stably in normal growth conditions with expression levels increasing with OD₆₀₀ values throughout log phase. The expression level of STP17 reaches a peak value at an OD₆₀₀ value of 1.2. Then, expression levels decrease with OD₆₀₀ values at stationary phase.

To investigate the role of the stp17 gene in S. Typhi, the stp17 mutant was constructed by homologous recombination mediated by the suicide plasmid. We measured growth of the wild-type and Δstp17 strain and found that growth of stp17 mutant strain was significantly slower compared to wild type (P < 0.05; Figure 3A). In addition, the growth of complementary strain Δstp17(pACYC184-stp17) restored to the wild type level (Figure 3A). At the same time, we constructed the S. Typhi stp17 over-expression strain wt(pBAD-stp17) and found that cell growth upon STP17 over-expression was significantly increased compared with the wild type strain (P < 0.05; Figure 3B). As shown in Figure 3C, the growth of pBSSB1-deficient strain ΔpBSSB1 was obviously decreased compared with the wild type strain because stp17 was absent due to the loss of pBSSB1. However, the growth rate of stp17 complementary ΔpBSSB1 strain ΔpBSSB1(pACYC184-stp17) was similar to the wild type strain. In addition, we compared the stability of pBSSB1 in wild type and Δstp17, and found no obvious difference presenting after deletion of stp17 (data not shown). These results suggest that stp17 has an effect on S. Typhi cell growth in a pBSSB1 independent manner.

As shown in Figure 4A, the motility of Δstp17 was greatly decreased compared with the wild type strain, and the motility of pBSSB1-deficient strain ΔpBSSB1 was obviously decreased as well because stp17 was absent due to the loss of pBSSB1. However, bacterial motility was restored obviously in the complementary strain Δstp17(pACYC184-stp17) and ΔpBSSB1(pACYC184-stp17). Moreover, the motility of stp17 over-expression strain wt(pBAD-stp17) was much greater compared with the control strain wt(pBAD) (Figure 4C). The ring diameters for various strains are shown in Figures 4B,D. Moreover, the expression of flagellar gene fljB^z66 and fljC show no obvious difference when stp17 was deleted (data not shown). This result suggests that the differences of bacterial motility may be not due to the different expression of flagellar gene. The data demonstrate that stp17 affects the motility of S. Typhi in a pBSSB1 independent manner.

The structure of STP17 was predicted by I-TASSER online server, as explained in Materials and Methods. As shown in Figure 5A, the 213 amino acids of STP17 contains nine Helix segments, six Stand segments and some Coil segments in the secondary structural level. The top-ranked 3-D structure of STP17 predicted by I-TASSER was shown in Figure 5B, and it contains a putative ATPase domain like a pocket (Figure 5C), with the top-ranked predicted residues GLY16, GLY17, GLY19, LYS20, ASN133, ARG134, LYS157, and LYS158 as ATPase active site residues (Figure 5A).

To define the active site of STP17 activity, eight mutants of site-directed mutagenesis were generated, and their growth and motility were compared to the wild type strain. As shown in Figure 6, when the residues of GLY16, GLY17, GLY19, LYS20, ASN133, ARG134, LYS157, and LYS158 mentioned above were mutated, the growth and motility of these mutant strains Δstp17::pACYC184-stp17^G16A, Δstp17::pACYC184-stp17^G19A,_Δstp17::pACYC184- stp17^K20A,_ Δstp17::pACYC184- stp17^N133A,_Δstp17::pACYC184- stp17^K157A, and Δstp17::pACYC184-stp17^K158A were significantly decreased compared with that of wild type strain, while there was no obviously change in growth and motility of mutant strains Δstp17::pACYC184-stp17^G17A and Δstp17::pACYC184-stp17^R134A. In addition, the stability of wild type strain and above mutants shows no significant difference (data not shown). All these results suggest that the amino acid residues of GLY16, GLY19, LYS20, ASN133, LYS157, and LYS158 may be the active site residues of STP17.

To identify the potential ATPase activity of STP17, we investigated whether the ATP concentration in solution could be decreased in the presence of STP17. As shown in Figure 7, the concentration of ATP in solution decreased markedly after incubation with STP17 for 10 min and continued to decrease after incubation for 30- and 50-min, while the concentration of ATP in solution containing the control protein BSA which lacks the ATPase activity did not decreased. The reduction in ATP concentration indicates catalytic activity of STP17, suggesting that STP17 can catalyze ATP into ADP and free phosphate.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.