The highly conserved TldD and TldE proteins of Escherichia coli are involved in microcin B17 processing and in CcdA degradation

Abstract

Microcin B17 (MccB17) is a peptide antibiotic produced by Escherichia coli strains carrying the pMccB17 plasmid. MccB17 is synthesized as a precursor containing an amino-terminal leader peptide that is cleaved during maturation. Maturation requires the product of the chromosomal tldE (pmbA) gene. Mature microcin is exported across the cytoplasmic membrane by a dedicated ABC transporter. In sensitive cells, MccB17 targets the essential topoisomerase II DNA gyrase. Independently, tldE as well as tldD mutants were isolated as being resistant to CcdB, another natural poison of gyrase encoded by the ccd poison-antidote system of plasmid F. This led to the idea that TldD and TldE could regulate gyrase function. We present in vivo evidence supporting the hypothesis that TldD and TldE have proteolytic activity. We show that in bacterial mutants devoid of either TldD or TldE activity, the MccB17 precursor accumulates and is not exported. Similarly, in the ccd system, we found that TldD and TldE are involved in CcdA and CcdA41 antidote degradation rather than being involved in the CcdB resistance mechanism. Interestingly, sequence database comparisons revealed that these two proteins have homologues in eubacteria and archaebacteria, suggesting a broader physiological role.

FIG. 1.

SOS induction triggered by an MccB17-producing plasmid (pCID909) in the tld deletion mutants. Induction of the SOS system was monitored using an sfiA::lacZ fusion. Isogenic strains CSH50 λsfiA::lacZ (strain 1) and its ΔtldD ΔtldE (strain 2), ΔtldD (strain 3), and ΔtldE (strain 4) mutants containing the plasmids indicated in the figure were streaked on MacConkey plates containing 1% lactose. The picture was taken after overnight incubation at 37°C. Plate A, the four strains carried the pACYC184 control vector. Plate B, the four strains carried the pCID909 plasmid. Plate C, the four strains carried the pKK-tldD and pCID909 plasmids. Plate D, the four strains carried the pKK-tldE and pCID909 plasmids.

FIG. 2.

Lack of lysis around tld deletion mutants. Lysis tests were carried out by stabbing individual colonies on a lawn of sensitive bacteria (C600). The CSH50λsfiA::lacZ and isogenic tld mutants and the plasmid they contained (pACYC184 or pCID909) are indicated.

FIG. 3.

Accumulation of pro-MccB17 in tld deletion mutants. Cultures were labeled with [³⁵S]cysteine (upper panel) or [¹⁴C]leucine (lower panel), and total cell extracts were prepared as described in Materials and Methods. The position of the 6.5-kDa molecular mass marker is indicated on the left and that of pro-MccB17 is on the right. The strains are derivatives of CSH50λsfiA::lacZ carrying the MccB17-producing plasmid pCID909 or the pACYC184 vector control. Lane 1, wild type/pACYC184; lane 2, wild type/pCID909; lane 3, ΔtldD ΔtldE/pACYC184; lane 4, ΔtldD ΔtldE/pCID909.

FIG. 4.

Mature form of MccB17 is detected in supernatants of wild-type strain cultures but not in those of tld deletion mutant cultures. Cultures were labeled with [³⁵S]cysteine, and supernatant samples were prepared as described in Materials and Methods. Lane M, molecular mass markers; lane 1, wild type/pACYC184; lane 2, wild type/pCID909; lane 3, ΔtldD ΔtldE/pACYC184; lane 4, ΔtldD ΔtldE/pCID909. Mature MccB17 is indicated by an arrow.

FIG. 5.

Arabinose-dependent CcdA41 expression prevents CcdB-mediated killing. Strain SG22622 and its derivatives, containing pULB3571 (pBAD-ccdA41) and pULB2232 (pACYC184 expressing CcdB), were streaked on LB plates containing the appropriate antibiotics and arabinose at the indicated concentration. The picture was taken after overnight incubation at 37°C. Strain genotypes are as follows: 1, gyrA462; 2, wild type; 3, ΔtldD; 4, ΔtldE; and 5, ΔtldD ΔtldE.

FIG. 6.

Western blot analysis of CcdA41 and CcdA. (A) Analysis of CcdA41 and CcdB produced by the wild-type and by the tld deletion mutants. SG22622 and its derivatives containing pULB2208 (ptac-ccdA41-ccdB) were grown in LB medium containing the appropriate antibiotic and 0.5 mM IPTG. At an OD₆₀₀ of 0.2 to 0.3, 1-ml samples were collected and analyzed by Western blotting with anti-CcdA (upper panel) or anti-CcdB (lower panel) antibodies. Strain genotypes are as follows: 1, wild type; 2, ΔtldD; 3, ΔtldE; 4, ΔtldD ΔtldE; 5, Δlon; and 6, Δlon ΔtldD ΔtldE. (B) Analysis of CcdA stability in a tld deletion mutant. SG22622 and its derivatives (Δlon and ΔtldD ΔtldE) containing pULB3565 (ptac-ccdA) were grown in LB containing the appropriate antibiotic to an OD₆₀₀ of 0.2 to 0.3. IPTG (0.5 mM) was added to induce CcdA expression for an hour. Spectinomycin was then added (200 μg per ml of culture) to block protein synthesis, and 1-ml samples were collected at the times indicated. Samples were treated and analyzed by Western blotting with anti-CcdA antibodies.