Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms

Transition metals are toxic at high concentrations. The P1B-ATPase metal exporter CtpC/Rv3270 is required for resistance to zinc poisoning in the human pathogen Mycobacterium tuberculosis. Here, we discovered that zinc resistance also depends on the chaperone-like protein PacL1/Rv3269. PacL1 bound Zn2+, but unlike PacL1 and CtpC, the PacL1 metal-binding motif (MBM) was required only at high zinc concentrations. PacL1 co-localized with CtpC in dynamic microdomains within the mycobacterial plasma membrane. Microdomain formation did not require flotillins nor the PacL1 MBM. Instead, loss of the PacL1 Glutamine/Alanine repeats led to loss of CtpC and sensitivity to zinc. PacL1 and CtpC are within the same operon, and homologous PacL1-P1B-ATPase pairs are widely distributed within and across prokaryotes. PacL1 colocalized and functioned redundantly with PacL orthologs in Mycobacterium tuberculosis. Overall, our study suggests that PacL proteins are scaffolds that assemble P-ATPase-containing metal efflux platforms, a novel type of functional membrane microdomain that underlies bacterial resistance to metal poisoning.

Genome Sequence of Brevundimonas sp., an Arsenic Resistant Soil Bacterium

Brevundimonas sp. is a bacteria able to grow in metal(loid) contaminated soil from Puchuncaví Valley, central Chile. This study has isolated a bacterial strain capable of growth under high doses of arsenic (As) (6000 mg L−1), and a draft genome sequence was generated. Additionally, real-time PCR was performed to examine the effect of As on some genes related to As resistance. Results demonstrated a total of 3275 predicted annotated genes with several genes related to the ars operon, metal(loid) resistance-related genes, metal efflux pumps, and detoxifying enzymes. Real-time PCR showed that the arsB involved in the efflux of As was down-regulated, whereas arsR, arsH, and ACR3 did not show differences with the addition of As. Our study provides novel evidence of diverse As regulating systems in tolerant bacteria that will lead to a better understanding of how microorganisms overcome toxic elements and colonize As contaminated soils and to the possible use of their specific properties in bioremediation.

Cryo-EM Structures of CusA Reveal a Mechanism of Metal-Ion Export

ABSTRACT Gram-negative bacteria utilize the resistance-nodulation-cell division (RND) superfamily of efflux pumps to expel a variety of toxic compounds from the cell. The Escherichia coli CusA membrane protein, which recognizes and extrudes biocidal Cu(I) and Ag(I) ions, belongs to the heavy-metal efflux (HME) subfamily of RND efflux pumps. We here report four structures of the trimeric CusA heavy-metal efflux pump in the presence of Cu(I) using single-particle cryo-electron microscopy (cryo-EM). We discover that different CusA protomers within the trimer are able to bind Cu(I) ions simultaneously. Our structural data combined with molecular dynamics (MD) simulations allow us to propose a mechanism for ion transport where each CusA protomer functions independently within the trimer. IMPORTANCE The bacterial RND superfamily of efflux pumps mediate resistance to a variety of biocides, including Cu(I) and Ag(I) ions. Here we report four cryo-EM structures of the trimeric CusA pump in the presence of Cu(I). Combined with MD simulations, our data indicate that each CusA protomer within the trimer recognizes and extrudes Cu(I) independently.

In silico identification and characterization of sensory motifs in the transcriptional regulators of the ArsR-SmtB family

The ArsR-SmtB family of transcriptional repressors regulates the transcription of metal-efflux proteins by binding specific metals at a variety of secondary structural elements, called motifs, on the surface of the proteins.