Predicted 3-D structures and conserved domains of the HK C5041 and the RR C5040.

<p>The exact positions of each domain are described in the text. 3-D structures were predicted by I-TASSER online server. (A) HK C5041. The signature segments (H, N, G1, F, and G2) of the kinase domain (CA, blue) and dimerization and histidine phosphorylation domain (DHp, yellow and purple) were identified by sequence alignment and indicated. The conserved histidine residue is indicated by green. The two <u>t</u>rans<u>m</u>embrane <u>h</u>elices (TMH-1 and TMH-2) were predicted by transmembrane protein topology prediction tool TMMOD. (B) RR C5040. The receiver domain (yellow) containing the putative aspartate residue (Asp, purple) for phosphorylation during signaling cascade, the AAA⁺ ATPase domain (blue), and the helix-turn-helix (HTH, red) domain for DNA binding are shown.</p

Oxygen tension modulates the expression of two operons in genomic island via TCS C5041/C5040.

<p>(A) Target genes' expression in <i>c5041</i>/<i>c5040</i> mutants under aerobic and anaerobic conditions. The expression of <i>c5032</i>-<i>lacZ</i> or <i>c5038</i>-<i>lacZ</i> in WT and Δ<i>c5041</i>/<i>c5040</i> mutant (LMP108) under aerobic and anaerobic conditions were compared. (B) Induction of <i>c5041</i>/<i>c5040</i> expression by oxygen deficiency. <i>c5041</i>-<i>lacZ</i> and <i>c5040</i>-<i>lacZ</i> transcriptional fusion strains were grown in M9 medium containing α-KG as the sole carbon source under aerobic and anaerobic conditions, respectively. β-galactosidase activity was measured and the values shown are means plus standard deviations of triplicate samples.</p

<i>c5032</i>-<i>5039</i> genes are located on a genomic island and contribute to UPEC fitness <i>in vivo</i>.

<p>(A) Similar genomic regions of <i>E. coli</i> K12 strain MG1655, EHEC strain EDL933, and UPEC strain CFT073 were compared and it was found that <i>c5032</i> to <i>c5041</i> genes are inserted between <i>tyrB</i> and <i>aphA</i> genes and can be considered as a genomic island. Within the island, three predicted operons shown as white, grey, and black arrows, respectively. Picture was drawn to scale. UPEC, uropathogenic <i>E. coli</i>; EHEC, enterohemorrhagic <i>E. coli</i>. (B) Island genes <i>c5032</i>-<i>5039</i> contribute to UPEC fitness <i>in vivo</i>. An <i>in vivo</i> competition assay was used. Equally mixed WT and Δ<i>c5032</i>-<i>5039</i>::Chl^r mutant bacteria containing approximately 2×10⁹ CFU were transurethrally inoculated into female mice. Two days after infection, the mice were sacrificed and their bladders and kidneys were aseptically removed. Wild-type and mutant bacteria were recovered by plating homogenized tissue samples on LB or LB containing chloramphenicol and their viable counts were determined. Each dot represents log₁₀CFU/g in the bladder or kidney from an individual animal and the detection limit is 1000 CFU/g. Bars indicate the median log₁₀CFU/g. A two-tailed Wilcoxon matched pairs test was performed, and the difference in colonization levels of WT and mutants was considered statistically significant if <i>P</i><0.05.</p

Contribution of <i>c5032</i>-<i>5039</i> to growth on α-KG and induction of <i>c5032</i>-<i>lacZ</i> or <i>c5038</i>-<i>lacZ</i> expression by α-KG.

<p>(A) <i>In vitro</i> growth of island gene mutants in M9 medium containing α-KG as the sole carbon and energy source. Optical density of the CFT073 WT and mutants during growth in M9 medium containing α-KG as the sole carbon source under anaerobic conditions was determined. Growth bars represent the average measurement at each time point from triplicate experiments. (B) Effects of different substrates on <i>c5032</i>-<i>lacZ</i> or <i>c5038</i>-<i>lacZ</i> expression. Gluc, glucose; Glyc, glycerol; TMAO, Trimethylamine <i>N</i>-oxide. (C) The induction of <i>c5038</i>-<i>lacZ</i> expression by α-KG is dose-dependent. Bacteria were grown in M9 medium containing 0.25% glycerol as the sole carbon source plus different amounts of α-KG as stimulus under anaerobic conditions.</p

Overexpression of <i>c5040</i> upregulated <i>c5035</i> expression independent of the natural stimulus.

<p>Bacteria were grown in human urine under aerobic conditions before being subjected to RNA extraction. qRT-PCR expression values are means plus standard deviations (error bars) of three independent experiments. (A) <i>c5035</i> expression in various TCS mutants compared to WT. (B) <i>c5040</i> expression in Δ<i>c5041</i> and Δ<i>c5041</i>::Chl^r mutants compared to WT. (C) <i>c5035</i> expression in Δ<i>c5041</i> mutant carrying the pMAL plasmid with <i>c5040</i> under the control of P_tac promoter as compared to Δ<i>c5041</i> mutant carrying the empty vector (e.v.). IPTG is present at 1 mM for induction.</p

Deletion of <i>c5041</i>/<i>c5040</i> reduces the colonization of the murine urinary tract by UPEC strain CFT073.

<p>(A) The WT and Δ<i>c5041</i>/<i>c5040</i>::Chl^r mutant strains were mixed to a 1∶1 ratio and approximately 2×10⁹ CFU were transurethrally inoculated into female mice. Two days after infection, the mice were sacrificed and their bladders and kidneys were aseptically removed. WT and mutant bacteria were recovered by plating homogenized tissue samples on LB or LB containing chloramphenicol and their viable counts were determined. (B) and (C), for the <i>in vivo</i> complementation assay, a mixture of wild-type strain containing empty vector (e.v.) pGEN-MCS and mutant strain containing empty vector or wild-type strain containing empty vector and mutant strain containing complementation plasmid (p<i>c5041</i>/<i>c5040</i>) were incolulated to female mice as in (A). Homogenized tissue samples (B, bladder; C, kidneys) were plated on LB with ampicillin or LB with both ampicillin and chloramphenicol. Each dot represents log₁₀CFU/g in the bladder or kidney from an individual animal and the detection limit is 1000 CFU/g. Bars indicate the median log₁₀CFU/g. A two-tailed Wilcoxon matched pairs test was performed, and the difference in colonization levels of WT and mutants was considered statistically significant if P<0.05.</p

A schematic model for the regulatory mechanism of C5041/C5040 and its controlled target genes.

<p>In certain host environments of UPEC, such as the epithelial cells of renal proximal tubule, where oxygen tension is low and α-KG is abundant, KguS/KguR can sense and respond to these stimuli by activating the expression of <i>c5038</i> encoding dicarboxylate transporter and <i>c5032</i>-<i>5037</i> genes encoding metabolic enzymes, involved in the utilization of α-KG as carbon and energy source. Possession and functionality of this system may meet special metabolic needs for UPEC during urinary tract infections. Question mark indicates function is unknown. Blue dots, α-KG (α-ketoglutarate); Succ-CoA, succinyl-CoA; Succ, succinate.</p

Differentially expressed proteins identified in mutant strains of UPEC CFT073 as compared to the wild-type when cultured in human urine.

a<p>Molecular weight.</p>b<p>Isoelectric point.</p>c<p>Confidence interval (Ion Confidence Interval % greater than 95 were considered significant).</p>d<p>Average from two biological repeats.</p

The role of <i>c5040</i>.

<p>(A) Effects of <i>c5040</i> on the expression of target genes in response to α-KG. Bacteria were anaerobically grown in M9 medium containing glycerol as sole carbon source and TMAO as electron acceptor in the absence or presence of α-KG. The expression of <i>c5032</i>-<i>lacZ</i> or <i>c5038</i>-<i>lacZ</i> in WT, Δ<i>c5040</i> (LMP107), and the complemented strains were represented by the β-galactosidase activity. (B) Non-radioactive EMSA studying the binding of MBP-C5040-His₆ to the promoter regions. PCR products of <i>c5032</i> promoter region (left panel) and <i>c5038</i> promoter region (right panel) were used as probes. Purified MBP-C5040-His₆ fusion protein was added in different concentration in each reaction mixture as indicated and MBP-His₆ was used as negative control at 300 ng per each reaction. For the lane on the right on each panel, a negative control DNA fragment amplified from coding region of <i>c5036</i> was used. DNA fragments were stained with SYBR green. (C) <i>In vitro</i> growth of Δ<i>c5040</i> mutants. Optical density of the CFT073 WT, Δ<i>c5040</i> mutant, and the complemented strain during growth in M9 medium containing α-KG as the sole carbon source under anaerobic conditions was measured. Growth bars represent the average measurement at each time point from triplicate experiments.</p