Copper Efflux Is Induced during Anaerobic Amino Acid Limitation in Escherichia coli To Protect Iron-Sulfur Cluster Enzymes and Biogenesis

Abstract

bAdaptation to changing environments is essential to bacterial physiology. Here we report a unique role of the copper homeostasis system in adapting Escherichia coli to its host-relevant environment of anaerobiosis coupled with amino acid limitation. We found that expression of the copper/silver efflux pump CusCFBA was significantly upregulated during anaerobic amino acid limitation in E. coli without the supplement of exogenous copper. Inductively coupled plasma mass spectrometry analysis of the total intracellular copper content combined with transcriptional assay of the P cusC -lacZ reporter in the presence of specific Cu(I) chelators indicated that anaerobic amino acid limitation led to the accumulation of free Cu(I) in the periplasmic space of E. coli, resulting in Cu(I) toxicity. Cells lacking cusCFBA and another copper transporter, copA, under this condition displayed growth defects and reduced ATP production during fumarate respiration. Ectopic expression of the Fe-S cluster enzyme fumarate reductase (Frd), or supplementation with amino acids whose biosynthesis involves Fe-S cluster enzymes, rescued the poor growth of ⌬cusC cells. Yet, Cu(I) treatment did not impair the Frd activity in vitro. Further studies revealed that the alternative Fe-S cluster biogenesis system Suf was induced during the anaerobic amino acid limitation, and ⌬cusC enhanced this upregulation, indicating the impairment of the Fe-S cluster assembly machinery and the increased Fe-S cluster demands under this condition. Taken together, we conclude that the copper efflux system CusCFBA is induced during anaerobic amino acid limitation to protect Fe-S cluster enzymes and biogenesis from the endogenously originated Cu(I) toxicity, thus facilitating the physiological adaptation of E. coli.

Interplay between tolerance mechanisms to copper and acid stress in Escherichia coli

“…S12). These results suggested that Cu stress in our experimental conditions interfered with the synthesis, maturation, and incorporation of Fe-S clusters into enzymes via the Isc pathway as proposed previously (13,14,20). However, contrary to these prior reports, the amounts of Cu used in these experiments were not sufficient to induce expression of sufA and sufB, two genes that encode components of the Suf pathway for Fe-S cluster repair (SI Appendix, Fig.…”

“…S9C), likely because these thiol-containing molecules are high-affinity chelators of Cu. We also tested branched-chain amino acids (BCAAs, i.e., Ile, Leu, and Val), which are known to rescue Cu-sensitive mutants of E. coli from Cu stress at pH 7 (13,14). These amino acids rescued growth of the ΔcopA mutant strongly at pH 7 ( Fig.…”

“…Several proteins that contain Fe (particularly Fe-S clusters) (13,14,21,26,27), Zn (28), and Mn (29) have now been identified as targets of Cu poisoning. Because metalloproteins account for nearly half of all enzymes in cells (30), precisely which enzymes are mismetallated by Cu and the ensuing changes in bacterial physiology may vary, depending on the specific organism and experimental conditions.…”

Bacterial copper storage proteins

“…A role for these proteins in Cu(I) storage is currently the most logical suggestion for their function, but in many cases what they are storing Cu for remains unknown. The presence of bacterial Cu storage proteins seems consistent with a number of other observations: (1) that bacterial Cu-import systems exist (6,7,17,21,52,56,77,95), including into the cytosol; (2) that endogenous pools of the metal are available in bacteria (11,15,16,18,96); and (3) that E. coli grown in both LB and minimal medium accumulates Cu (97). It also highlights that there are alternative mechanisms to using different cellular compartments to prevent mis-metallation of proteins by Cu (37).…”

“…Copper availability appears to be largely constrained by the use of high affinity sites in proteins (12)(13)(14), although 'pools' of Cu bound by other molecules are important (4,5,11,(15)(16)(17)(18).…”