“Role of the two ClpP protease subunits in Mycobacterium tuberculosis”

PhDCaseinolytic (Clp) proteases are the most widespread energy-dependent proteases in bacteria. They are involved in protein quality control by degrading misfolded and aggregated proteins and have a role in regulatory proteolysis. The main group of substrates of the Clp proteases is the SsrA-tagged proteins, which arise in the presence of defective translation. SsrA tagging is carried out by tmRNA, encoded by ssrA, together with a protein partner SmpB. While most organisms have only one ClpP, Mycobacterium tuberculosis has two ClpP protease subunits (ClpP1 and ClpP2) with at least one of them essential for growth. Co-expression of clpP1 and clpP2 was demonstrated showing that clpP1 and clpP2 are not expressed under different conditions. The promoter region of clpP1P2 was identified, together with the potential ClgR binding site. A reporter system to assay ClpP1 and ClpP2 enzymatic activities was developed based on LacZ incorporating SsrA tag sequences. This showed that both ClpP1 and ClpP2 degrade SsrA-tagged LacZ, whilst only ClpP2 degrades untagged proteins. This suggests different pattern recognition for the two ClpP proteins with substrate recognition by ClpP1 dependent on the last three residues of the C-terminus of the tag sequence. Mutagenesis analysis of the accessory components demonstrated that ssrA is essential but SmpB deletion is viable. SmpB is not required for aerobic growth but the smpBΔ mutant strain was more sensitive to antibiotics targeting the ribosome as compared to wildtype cells

御用留記（天保十三壬寅年十一月一日より三十日迄、益子信彭）

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated cas genes are sequence-specific DNA nuclease systems found in bacteria and archaea. CRISPR/Cas systems use RNA transcripts of previously acquired DNA (spacers) to target invading genetic elements with the same sequence, including plasmids. In this research we studied the relationship between CRISPR/Cas systems and multidrug resistance in Escherichia coli . The presence of Type I-E and Type I-F CRISPR systems was investigated among 82 antimicrobial-susceptible and 96 MDR clinical E. coli isolates by PCR and DNA sequencing. Phylogrouping and MLST were performed to determine relatedness of isolates. RT-PCR was performed to ascertain the expression of associated cas genes. Type I-F CRISPR was associated with the B2 phylogroup and was significantly overrepresented in the susceptible group (22.0%) compared with the MDR group (2.1%). The majority of CRISPR I-F-containing isolates had spacer sequences that matched IncF and IncI plasmids. RT-PCR demonstrated that Type I-F cas genes were expressed and therefore potentially functional. The CRISPR I-F system is more likely to be found in antimicrobial-susceptible E. coli . Given that the Type I-F system is expressed in WT isolates, we suggest that this difference could be due to the CRISPR system potentially interfering with the acquisition of antimicrobial resistance plasmids, maintaining susceptibility in these isolates

Mycobacterium tuberculosis ClpP Proteases Are Co-transcribed but Exhibit Different Substrate Specificities

PMCID: PMC3613350This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Identification of key residues in a regulatory region of the <i>clpP1P2</i> operon.

<p>A) Sequence of the region upstream of <i>clpP1P2</i>. The <i>tig</i> stop codon, −10 promoter element, and <i>clpP1</i> start codons are underlined. The 18 nucleotides that constitute the regulatory region are boxed in grey. The CGC region mutated is underlined in bold. B) Identification of a regulatory region. The CGC motif (underlined bold) was mutated to AAA. C) Mapping of a regulatory binding site. Single nucleotide substitutions in P₂₇₈ were made by SDM. Residues A or T were mutated to G or and residues C or G were mutated to A. Results are the average activity of three independent transformants assayed in duplicate ± standard deviation. Activity is given in Miller Units- measured as nmol of O-nitrophenol produced per min per mg of protein. A significant difference of activity compared to wild-type P₂₇₈ is marked by an *(p<0.05) using the student’s t-test (unpaired, two sided).</p

P₂₇₈ promoter activity was not induced by stress treatments.

<p>A) P₂₇₈ promoter activity in standing liquid cultures. Treatments were: 50 µg/mL of chlorpromazine for 3 h, 10 µg/mL of menadione for 3 h, 10 µg/mL of valinomycin for 3h, 6 µg/mL of vancomycin for 90 min. B) Promoter activity in rolling cultures. Treatments were: 42°C for 1 h, 10 mM diamide for 1 h C) Promoter activity in response to diamide treatment in <i>M. tuberculosis</i> CDC1551. D) Promoter activity in response to diamide and vancomycin treatments in presence or absence of streptomycin selection. Stress treatments were 10 mM diamide for 1 h or 6 µg/mL of vancomycin for 90 min. The average and standard deviation of three independent transformants assayed in duplicate is given. ß-galactosidase activity is given in Miller Units - measured as nmol of O-nitrophenol produced over time (min) per mg of protein. Activity is given in Miller Units (MU)- measured as nmol of O-nitrophenol produced per min per mg of protein. The background activity from pSM128 (control vector) was 6±3 MU under the different conditions tested.</p

<i>clpP1</i> and <i>clpP2</i> are co-transcribed as an operon.

<p>A) Chromosomal organisation of <i>clpP1</i> and <i>clpP2</i>. Regions amplified for RT-PCR are marked. B) Limiting dilution semi-quantitiative RT-PCR. RNA was extracted from <i>M. tuberculosis</i> grown to late exponential phase in liquid cultures and cDNA was synthesised from 1 µg of RNA. Serial 4- fold dilutions of cDNA were used as a template for PCR using primers specific for <i>clpP1</i> (P1), <i>clpP2</i> (P2), the <i>clpP1</i>-<i>clpP2</i> junction (P1P2) and <i>sigA</i>. C: no RT control; B: no template blank, M: markers.</p

Identification of the promoter of the <i>clpP1P2</i> operon.

<p>A) The regions upstream of <i>clpP1</i> or <i>clpP2</i> tested for promoter activity are marked. B) P₁₂₅ activity in <i>M. tuberculosis</i>. C) P<i>_clpP2</i> activity in <i>M. tuberculosis</i>. Promoter activity was measured in transformants grown to late exponential phase in standing liquid cultures. Results are the average activity ± standard deviation of three independent transformants assayed in duplicate. Activity is given in Miller Units (MU)- measured as nmol of O-nitrophenol produced per min per mg of protein. Control = pSM128 empty vector control.</p

Identification of the −10 promoter element.

<p>A) Sequence of the P₁₂₅ region upstream of <i>clpP1P2.</i> Putative −10 elements (10A and 10B) are boxed. The residues mutated are in bold. The predicted ClpP1 start codon and Tig stop codons are indicated. B) Identification of the −10 element. The following mutations were made - 10A: TAGTGT mutated to <b>C</b>AGTG<b>G</b>; 10B: TAGAAG mutated to <b>CG</b>GAAG. Results are the average activity of three independent transformants assayed in duplicate ± standard deviation. Activity is given in Miller Units (MU)- measured as nmol of O-nitrophenol produced per min per mg of protein. The background activity from pSM128 (control vector) was 4±2 MU. A significant difference, measured by the student’s t-test (unpaired, two sided), compared to the control vector (pSM128) is marked by an *(p<0.05).</p

Promoter activity during aerobic growth, hypoxia, and reaeration in <i>M. tuberculosis.</i>

<p>A) <i>M. tuberculosis</i> transformants harbouring P₂₇₈ were grown in aerobic culture. Results are the average activity of three transformants against average OD₅₈₀. A significant difference, measured by the student’s t-test (unpaired, two sided), compared to promoter activity at OD₅₈₀ = 0.15 is marked by an *(p<0.05). B) P₂₇₈ promoter activity in the Wayne model of hypoxia. <i>M. tuberculosis</i> liquid cultures were inoculated to a theoretical starting OD₅₈₀ of 0.004 in DTA medium. A significant difference compared to activity at day 0 is marked by an *(p<0.05) using the student’s t-test (unpaired, two sided). C) P₂₇₈ promoter activity after reaeration. Long term hypoxic cultures (12 weeks) were used to inoculate medium and grown in aerobic rolling cultures. Cell-free extracts were prepared once the cultures reached an OD₅₈₀ of 0.3. Results are the average activity of three independent transformants assayed in duplicate ± standard deviation. Activity is given in Miller Units- measured as nmol of O-nitrophenol produced per min per mg of protein.</p

Protein turnover in strains over-expressing ClpP subunits.

<p>A to D) <i>M. tuberculosis</i> transformants were grown to late exponential phase in standing liquid cultures in presence of succinate +/− acetamide (0.1% w/v) and cell-free extracts were prepared and β-galactosidase activity measured. Empty bars: uninduced conditions (succinate); Grey striped bars: induced conditions (succinate+acetamide). A significant difference measured by the student’s t-test (unpaired, two sided) compared to the induced LacZ level in the WT strain is marked by an *(p<0.05). E) Three <i>M. tuberculosis</i> transformants carrying LacZ-ASV were grown to late exponential phase in standing liquid cultures in presence of acetamide (0.1% w/v) and cell-free extracts were prepared. Treatments were 10 mM diamide for 1 h or 6 µg/mL of vancomycin for 90 min. A significant difference from untreated WT is marked by an *(p<0.05). Results are the average activity of three independent transformants assayed in duplicate ± standard deviation. Activity is given in Miller Units- measured as nmol of O-nitrophenol produced per min per mg of protein. Strains are- WT: wild-type; P1: over-expressing ClpP1; P2: over-expressing ClpP2; P1P2: over-expressing ClpP1 and ClpP2.</p