As an IVI antigen identified in a previous study using IVIAT method, the regulation of YncD expression usually can be induced in certain conditions encountered in vivo. In the genome of S. Typhi, the yncD gene is adjacent to the yncE gene but it has the opposite transcriptional orientation. The yncE gene is induced under iron restriction through the action of the global iron regulator Fur in E. coli; however, the regulator and the iron restriction did not affect the transcription of the yncD gene (McHugh et al., 2003). Upstream of the yncD gene, a possible PmrAB-box sequence, cattttcttaacttaat, was found, which indicated that the

expression of the yncD gene may be regulated by the PmrAB system (Marchal et al., selleck kinase inhibitor 2004). In agreement with this anticipation, acidic pH, a main activation signal of the PmrAB system, was proved to induce the expression of yncD gene in the present study. The acid

condition is an ecological niche that pathogens usually encounter in vivo. Enteric pathogens share an oral route of infection (Gorden & Small, 1993; Maurer et al., 2005). During the initial infection, enteric bacteria RGFP966 research buy encounter low pH stresses in the human digestive tract (Drasar et al., 1969). Successful colonization requires survival through the stomach at pH 1–2 or the intestinal tract at pH 2–7 (Dressman et al., 1990). The bacteria respond to low pH stresses by regulating gene expression, which maintains internal pH homeostasis (Gorden & Small, 1993). Moreover, low pH is an important inducing factor of virulence genes as well. Low pH enhances the expression of numerous virulence factors, such as the ToxR-ToxT virulence regulon in Vibrio cholerae (Behari et al., 2001) and the phoP-phoQ regulon of Salmonella enterica (Bearson et al., 1998). It also enhances expression of genes for flagellar Methisazone motility and catabolism (Maurer et al., 2005). Due to lack of information, the exact function of YncD remains unclear. However, our study showed that YncD plays a role

in the in vivo survival of S. Typhi. As the yncD gene knockout significantly reduces bacterial virulence and the attenuated strain shows an effective immunoprotection, the yncD gene is undoubtedly a good candidate gene for the construction of attenuated vaccine strains. This study was supported by the National Natural Science Foundation of China (Grant No. 30500435). We gratefully acknowledge Victor de Lorenzo of the Centro Nacional de Biotecnologia CSIC, Spain, for providing the Mini-Tn5 plasmid. K.X. and Z.C. contributed equally to this work. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

, 2001, 2003). FlhD by itself, independent of FlhC, has also been reported to regulate cell division in E. coli (Prüß

& Matsumura, 1996). Cells in flhD mutant cultures were observed to continue dividing for several generations after cells in the flhD+ parental culture had stopped growing and entered the stationary VX-765 mw phase. This work is frequently cited as evidence that FlhD regulates cell division (Kaper & Sperandio, 2005; Umehara et al., 2007; Cui et al., 2008; Hatt & Rather, 2008; Isalan et al., 2008); however, our data indicate that this is not the case. We re-examined the effects of flhD mutations on entry to the stationary phase and found that the previously observed phenotype is not due to the flhD locus. Here, we show that the difference in the final cell number is due to the thyA mutation in the parental flhD+ strain, which had apparently reverted in the flhD− mutant strain used in the study. When the strains being compared have the same thyA allele (wild type or mutant), flhD mutations have no effect on growth. The E. coli K-12 strains and phage used in this study are listed in Table 1. λWM7 (Mao & Siegele, 1998) is a derivative of λRS45 (Simons et al., 1987) that carries an operon fusion between the mcb operon promoter (positions

−344 to +79) and the lac operon. Strains lysogenic for λWM7 were isolated by infecting YK410 and YK4131 with λWM7 and screening survivors on medium containing X-Gal Panobinostat where lysogens form blue colonies. Monolysogens were identified by measuring β-galactosidase activity in several independent Y-27632 2HCl isolates of

each lysogen. Transductions with P1vir were performed as described by Miller (1972). Hfr mapping was performed as described (Singer et al., 1989) using the Hfr strains described in that paper as donors. To facilitate the exchange of flhD alleles, derivatives of YK410 (λPmcb-lacZ) and YK4131 (λPmcb-lacZ) were constructed that carry the linked uvrC279∷Tn10 mutation and retain their original flhD allele. These are strains DS507 and DS511, respectively, which were used as the donor strains in all subsequent strain constructions. Motility assays (described below) were used to determine whether transductants carried the wild type or the mutant flhD allele. Introduction of the uvrC279∷Tn10 mutation did not affect the expression of the Pmcb-lacZ fusion (Table 2 and data not shown). For β-galactosidase assays, cultures were grown in TB medium [1% Bacto tryptone, 0.5% NaCl (Arber et al., 1983)] supplemented with MgSO4 (10 mM), thymidine (10 μg mL−1), and thiamine (2 μg mL−1). For plates, 1.3% Bacto agar (Difco Laboratories) was included.

However, the absolute levels of tmRNA were at least an order of magnitude higher than the corresponding levels of pre-tmRNA. The ratio of tmRNA : pre-tmRNA was 38 : 1 before the addition of erythromycin. A comparison of tmRNA with rRNA demonstrated that mature tmRNA levels were 7.2 ± 0.5% of 23S rRNA gene levels, increasing to 32.8 ± 5.6% following 3-h incubation in 16 μg mL−1 erythromycin. Thus, mature tmRNA was one of the most abundant non-rRNA RNA species in M. smegmatis. Increased levels of pre-tmRNA and tmRNA were also found in M. bovis BCG (a representative of the Mycobacterium tuberculosis complex) incubated

for 24 h in the presence of streptomycin (Supporting Information, Fig. S1). To rule out the possibility that the real-time RT-qPCR analysis biased the analysis of tmRNA levels, RNA samples Bleomycin nmr were also analyzed by Northern blot (Fig. 3b); these RNA preparations had not previously been tested by real-time RT-qPCR. From the Northern blot analysis, exposure to 2 μg mL−1 erythromycin increased tmRNA levels 2.3-fold (Fig. 3c); this correlated exactly with the 2.3 ± 0.2-fold increase determined by RT-qPCR analysis. Thus, real-time RT-qPCR analysis was deemed equivalent to Northern analysis. The results described above suggested that the mycobacterial ssrA promoter (which drives tmRNA

synthesis) was upregulated in the presence of ribosome inhibitors. However, the changes in tmRNA levels could be explained by changes in the rate of tmRNA degradation. Following inhibition of RNA synthesis with 100 μg mL−1 rifabutin, the mature tmRNA half-life was 50 min, which did not change following 3-h exposure to 16 μg mL−1 erythromycin HDAC assay (slopes and intercepts of degradation vs. time lines were not significantly different; P=0.6). Thus, exposure to erythromycin did not lead to a change in tmRNA degradation. The activity of the ssrA promoter was assessed using plasmid pFPSSRA-1, which carried this promoter driving expression of GFP

as a transcriptional reporter. The cloned DNA spanned DNA ligase from 254 bp upstream from the ssrA gene (141 bp into the upstream gene, dmpA) through the first 178 bp of the ssrA gene. Mycobacterium smegmatis FPSSRA-1 (i.e. carrying plasmid pFPSSRA-1) showed constitutive high-level GFP fluorescence, which increased approximately twofold when the organisms were grown in the presence of 2 μg mL−1 erythromycin. This was consistent with the ssrA promoter being constitutively active and inducible with macrolides. However, as erythromycin inhibits protein synthesis, it was felt that using GFP fluorescence would underestimate promoter activity. To validate the assumption that GFP mRNA levels represented the output of the ssrA promoter and not the accumulation of a stable transcript, the rate of degradation of this mRNA species was determined in M. smegmatis FPSSRA-1. The half-life of the GFP mRNA was deemed to be 2.5 min, i.e.