This suggested the presence of a terminator or other regulatory sequence in the intergenic

region that modulated the expression of gluQ-rs. Figure 3 The transcription of the gluQ-rs gene is controlled by a termination stem loop. A) Schematic representation of the operon, the arrows indicate the position of each promoter identified by our bioinformatics analysis and experimentally determined by Kang and Craig, 1990 [22]. The putative ρ-independent terminator is represented by the stem loop symbol upstream of gluQ-rs gene. The horizontal bar represents the DNA region amplified and cloned into pQF50 (Table 1). The recombinant plasmids are described in Table 1. pVCDT does not have the dksA promoter but has the terminator. pVCPDT has the promoter region of dksA and the terminator upstream of gluQ-rs; therefore, it represents the genomic organization of the operon. pVCPD also has the promoter of dksA but lacks MK-8776 the terminator region. The size of each fragment is indicated. B) β-galactosidase activity of each protein extract obtained from the corresponding clone. The data represent the average of three experiments in triplicates and the Student MEK162 cost t test was used to compare the means between each clone with the

empty vector. *** p values <0.05 were considered statistically significant. Table 1 Bacterial strains and plasmids used in this work Bacterial strains or plasmid Characteristics Source or reference Shigella flexneri     S. flexneri 2457T Wild type strain Laboratory stock S. flexneri 2457T ΔgluQ-rs::kan Deletion mutant of gluQ-rs gene This work Escherichia coli     E. coli W3110 ΔgluQ-rs::kan Deletion mutant of gluQ-rs gene [10] DH5α F - ϕ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK-, mK+) phoA supE44 λ-thi-1 gyrA relA1 [24] BL21(DE3) F - ompT gal dcm lon hsdS B (r B - m B - ) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) Invitrogen Plasmids     pTZ57R/T bla, pMB1 ori, lacZ peptide, f1 phage ori Fermentas® pQF50 bla, pMB1

ori, lacZ gene without promoter [23] pET15c Empty vector, a modified version of pET15b This work pVCDT S. flexneri fragment from nucleotide +58a of dksA gene to beginning of gluQ-rs gene (+590) cloned into pQF50. Pair of primers used were PgluQF/PdksARCT. This work pVCPDT S. flexneri fragment from nucleotide −506 of dksA ioxilan gene to beginning of gluQ-rs gene (+590) cloned into pQF50. Pair of primers used were PdksAF/PdksARCT. This work pVCPDTMut S. flexneri fragment from nucleotide −506 of dksA gene to beginning of gluQ-rs gene (+590) cloned into pQF50, with the terminator mutated by the nucleotides indicated in Figure 4a. This work pVCPD S. flexneri fragment from nucleotide −506 of dksA gene to nucleotide +527 (end of dksA gene) cloned into pQF50. Pair of primers used were PdksAF/PdksARST. This work pATGGQRS S. flexneri gene from nucleotide +509 (stop codon of dksA) to nucleotide +1469 (last codon of gluQ-rs without stop codon). Pair of primers used were ATGGQRSF/ATGGQRSR.