Genetic organization of the <i>kdp</i> operons in various bacteria and overexpression of the <i>kdpF</i> gene in <i>M. bovis</i><b> BCG.</b>

<p><b>A.</b> Transcriptional organization of the <i>kdp</i> operon in <i>E. coli</i> and various mycobacteria. An intergenic region of 192 bp separates the <i>kdpDE</i> operon from the <i>kdpFABC</i> operon in <i>M. tuberculosis</i> and <i>M. bovis</i> BCG. Arrows indicate the direction of transcription. <b>B.</b> Expression of <i>kdpF</i> transcripts relatively to those of s<i>igA</i> gene was measured by qRT-PCR from <i>M. bovis</i> BCG strains grown in Sauton’s medium carrying the pMV261 vector or p<i>kdpF</i> plasmid. Results are expressed as means ± SD from three independent experiments (each performed in triplicate).</p

Growth of <i>M. bovis</i> BCG overexpressing <i>kdpF</i> in murine and human primary macrophages.

<p><b>A</b>. Kinetic of growth of <i>M. bovis</i> BCG overexpressing <i>kdpF</i> in murine BMDM over a 8-day period. <b>B.</b> Bacterial number upon infection of HMDM infected with <i>M. bovis</i> BCG overexpressing <i>kdpF</i> at day 6 post-infection. The means ± SD calculated from three independent experiments (each performed in triplicate) are shown. Asterisks indicate statistical significance using a generalized mixed effects model (*** P<0.001).</p

Effect of <i>kdpF</i> overexpression on the expression of the <i>kdp</i> operons inside macrophages.

<p>RNA was extracted from <i>M. bovis</i> BCG overexpressing <i>kdpF</i> grown Sauton’s liquid medium or after 6 days infection of BMDM. Quantitative RT-PCR was used to study the expression of the <i>kdp</i> operon genes relative to the one of the <i>sigA</i> gene. Data are means ± SD calculated from three independent biological samples analyzed in triplicate. Statistical significance was performed using a generalized mixed effects model.</p

Morphotype of the KdpF-overexpressing strain.

<p><b>A.</b> Single <i>M. bovis</i> BCG colonies were grown on cord-reading agar and visualized after 3 weeks. Magnification is 16x for the main figures and 63x for the insets. <b>B.</b> SEM micrographs of single <i>M. bovis</i> BCG colonies grown in 7H10 agar plates at 6000× magnification. The inset micrographs are at 13740x and 12000× magnification for <i>M. bovis</i> BCG pMV261 and <i>M. bovis</i> BCG overexpressing <i>kdpF,</i> respectively.</p

<i>In vivo</i> interaction of KdpF with KdpD and effect of <i>kdpF</i> overexpression on the regulation of <i>kdp</i> genes by K⁺.

<p><b>A</b>. The interaction between KdpF and KdpD was assayed using the BACTH system by transforming <i>E. coli</i> BTH101 cells with KdpF-T25 and KdpD-T18 plasmids. Liquid β-galactosidase assays were performed from three independent experiments. Error bars represent standard deviations. <b>B.</b> K⁺-regulated expression of <i>kdp</i> operon. <i>M. bovis</i> BCG carrying the pMV261 vector or the p<i>kdpF</i> constructs were grown in Sauton’s medium or in K⁺-depleted Sauton’s medium. The levels of <i>kdpA, kdpB, kdpD</i> and <i>kdpE</i> transcripts relative to those of the <i>sigA</i> gene were measured by qRT-PCR. Results are expressed as means ± SD from three independent experiments (each performed in triplicate). Asterisks indicate statistical significance using a generalized mixed effects model (** P<0.01, *** P<0.001).</p

<i>In vivo</i> interaction of KdpF with MmpL7 variants using the BACTH system.

<p><b>A</b>. Predicted topology of MmpL7 using the topological analysis server TMHMM (<a href="http://www.cbs.dtu.dk/services/TMHMM-2.0/" target="_blank">http://www.cbs.dtu.dk/services/TMHMM-2.0/</a>). The TM regions are indicated by cylinders and are numbered while the two non-TM loops are indicated as Domain 1 and Domain 2. <b>B.</b> Interaction between KdpF-T25 and MmpL7-T18 constructs. <i>E.coli</i> BTH101 were co-transformed with <i>kdpF</i>-T25 plasmid and different constructs derived from pUT18 plasmid encoding MmpL7 full-length protein or portions of MmpL7 (TM 2–6, TM 8–12, cytoplasmic Domain 2). The portion coding for MmpL7 TM 8–12 was also cloned into pUT18c vector to obtain a reverse membrane orientation. The bars represent β-galactosidase activity expressed in Miller units ± SD of three independent experiments performed in triplicate.</p

Base-Promoted Expedient Access to Spiroisatins: Synthesis and Antitubercular Evaluation of 1<i>H</i>‑1,2,3-Triazole-Tethered Spiroisatin–Ferrocene and Isatin–Ferrocene Conjugates

The use of sodium hydride provides a convenient access to the synthesis of C-5-functionalized spiroisatins with the absence of the typical drawbacks associated with conventional protocols. The synthesized precursors, viz. <i>N</i>-alkylazido spiroisatins and their unprotected counterparts, were explored in Cu-mediated azide–alkyne cycloaddition reactions to probe the antitubercular structure–activity relationships (SAR) within the isatin–ferrocene–triazole conjugate family. The antitubercular evaluation studies of the synthesized conjugates revealed an improvement in the minimal inhibitory concentration (MIC) with the introduction of ferrocene nucleus, as evidenced by spiroisatin–ferrocene and isatin–ferrocene hybrids

Base-Promoted Expedient Access to Spiroisatins: Synthesis and Antitubercular Evaluation of 1<i>H</i>‑1,2,3-Triazole-Tethered Spiroisatin–Ferrocene and Isatin–Ferrocene Conjugates

The use of sodium hydride provides a convenient access to the synthesis of C-5-functionalized spiroisatins with the absence of the typical drawbacks associated with conventional protocols. The synthesized precursors, viz. <i>N</i>-alkylazido spiroisatins and their unprotected counterparts, were explored in Cu-mediated azide–alkyne cycloaddition reactions to probe the antitubercular structure–activity relationships (SAR) within the isatin–ferrocene–triazole conjugate family. The antitubercular evaluation studies of the synthesized conjugates revealed an improvement in the minimal inhibitory concentration (MIC) with the introduction of ferrocene nucleus, as evidenced by spiroisatin–ferrocene and isatin–ferrocene hybrids

Alignment of mycobacterial MgtC proteins and genetic environment of <i>mgtC</i> gene.

<p>(A) Alignment of <i>S.</i> Typhimurium MgtC (Accession Number AAL22622.1), <i>M. tuberculosis</i> MgtC (Accession Number NP_216327.1) and <i>M. marinum</i> MgtC (Accession Number ACC41130.1) using ClustalW. The upper line indicates the soluble C terminal part. Rectangles indicate four conserved residues that have been shown to be essential for <i>Salmonella</i> MgtC function. (B) Genetic environment of <i>mgtC</i> gene (striped arrows) in <i>M. tuberculosis</i> and <i>M. marinum</i> genomes. In both species, the <i>mgtC</i> gene is adjacent to <i>Rv1810</i> that is homologous to <i>MMAR_2686</i> (black arrows) and to PPE genes (grey arrows). The <i>MMAR_2688</i> gene is homologous to <i>Rv1812c</i>.</p

Expression of <i>Mma mgtC</i> and upstream genes in high Mg²⁺ and low Mg²⁺ conditions.

<p>(A) RT-PCR experiment on RNA isolated from <i>M. marinum</i> strains grown in high or low Mg²⁺ with primers specific for <i>mgtC</i>, <i>MMAR_2686</i>, <i>MMAR_2683</i> (PPE31) and <i>sigA</i>. Experiment was carried out with wild-type strain, <i>mgtC</i> mutant strain and complemented strain. Controls where reverse transcriptase was omitted (indicated by RT -) are done to verify the absence of genomic DNA contamination in the RNA sample. The <i>sigA</i> gene is used as control. (B) Quantification of <i>mgtC</i>, <i>MMAR_2686</i> and <i>MMAR_2683</i> RNA by Q-RT-PCR experiment using RNA isolated from <i>M. marinum</i> strains grown in high or low Mg²⁺. The sigma factor <i>sigA</i> was used as an internal standard. Results are expressed as means+standard deviations (SD) from a representative experiment performed in triplicate.</p