Thiolactomycin resistance in Escherichia coli is associated with the multidrug resistance efflux pump encoded by emrAB

Abstract

Thiolactomycin (TLM) and cerulenin are antibiotics that block Escherichia coli growth by inhibiting fatty acid biosynthesis at the 1-ketoacyl-acyl carrier protein synthase I step. Both TLM and cerulenin trigger the accumulation of intracellular malonyl-coenzyme A coincident with growth inhibition, and the overexpression of synthase I protein confers resistance to both antibiotics. Strain CDM5 was derived as a TLM-resistant mutant but remained sensitive to cerulenin. TLM neither induced malonyl-coenzyme A accumulation nor blocked fatty acid production in vivo; however, the fatty acid synthase activity in extracts from strain CDM5 was sensitive to TLM inhibition. The TLM resistance gene in strain CDM5 was mapped to 57.5 min of the chromosome and was an allele of the emrB gene. Disruption of the emrB gene converted strain CDM5 to a TLM-sensitive strain, and the overexpression of the emrAB operon conferred TLM resistance to sensitive strains. Thus, activation of the emr efflux pump is the mechanism for TLM resistance in strain CDM5.The 3-ketoacyl-acyl carrier protein (0-ketoacyl-ACP) synthases are key regulators of dissociated (type II) fatty acid synthase systems typified by Escherichia coli (for reviews, see references 6 and 16). P-Ketoacyl-ACP synthase I is required for a critical step in the elongation of unsaturated acyl-ACP, and fabB mutants lacking synthase I activity synthesize neither palmitoleic nor cis-vaccenic acids and require supplementation with unsaturated fatty acids for growth (29). 3-Ketoacyl-ACP synthase II is responsible for the temperature-dependent regulation of fatty acid composition (for a review, see reference 9). Mutants (fabF) lacking synthase II activity are deficient in cis-vaccenic acid but do not have a growth phenotype (12, 13). O-Ketoacyl-ACP synthase III selectively catalyzes the formation of acetoacetyl-ACP in vitro (19). Synthase III possesses acetyl coenzyme A (acetyl-CoA):ACP transacylase activity (34); however, it is unknown whether synthase III accounts for all of the acetyl transacylase activity. The role of this third condensing enzyme remains to be firmly established, but its position at the beginning of the biosynthetic pathway suggests that it plays a role in governing the rate of fatty acid initiation.Thiolactomycin [(4S)(2E,5E)-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene-4-thiolide] (TLM) is a unique antibiotic structure that inhibits type II (bacterial and plant) but not type I (Saccharomyces cerevisiae and mammalian) fatty acid synthases (14,15,23,24,26,27,31). The antibiotic is not toxic to mice and affords significant protection against urinary tract and intraperitoneal bacterial infections (23). Understanding the mechanism of TLM action is important to the development of more-effective antibiotics that exhibit selective action against type II bacterial fatty acid synthases. synthase suggests that the 3-ketoacyl-ACP synthase and the acetyl-CoA:ACP transacylase activities are the only individual enzymes inhibited by TLM in vitro (24). The findings that malonyl-ACP pr...

Molecular Properties of Bacterial Multidrug Transporters

“…ErmA, a 42.7-kDa membrane fusion protein with a single TMS and a large Cterminal periplasmic domain, is thought to form a continuous channel between the inner and outer membrane (126,135). EmrAB confers resistance to hydrophobic uncouplers and antibiotics (59,135). Tanabe et al (246) characterized the E. coli emrKY operon.…”

Inhibition of the fungal fatty acid synthase type I multienzyme complex

“…However, inhibition efficiency is also defined by other parameters, as, for example, the effective concentration in the cell interior. Indeed, for some organisms resistance toward the inhibitors TLM and CER was revealed to be caused by efflux pumps, which keep the inhibitor concentration below a critical level (44)(45)(46). In the case of FAS KS inhibition, the properties of the KS condensation mechanism and the compartmentalization of the type I fungal fatty acid synthase system might also affect inhibitor efficiency.…”

Malonate Decarboxylase in Bacteria and Its Application for Determination of Intracellular Acyl-CoA Thioesters

“…Malonyl-CoA is a minor component of the CoA pool in normally grown cells, but it dramatically increases to become the dominant component when the cells are treated with cerulenin or thiolactomycin 15,23) , antibiotics that inhibit b-keto-acyl ACP synthase and acetyl-CoA:ACP transacylase of fatty acid synthase 18,19,27,28,37,42,64,67,75,88,91) . This result suggests that malonyl-CoA does not exert negative feedback on acetyl-CoA carboxylase which produces malonylCoA wastefully in spite of blocking the sequence of fatty acid synthesis.…”

“…The enzymatic analysis is simple and sufficiently sensitive to detect pmol levels of the acyl-CoAs. The reaction mixture for the acyl-CoA cycling method contains one unit of malonate decarboxylase 12,15,16,23,65,84,86) , corresponding to approximately 300 pmol of the enzyme, i.e., 300 pmol of nonesterified ACP (d subunit). The free ACP is acylated by the e subunit in proportion to the amount of short chain acyl-CoAs given, the acyl residue of acyl-S-ACP formed is replaced with malonate to generate malonyl-S-ACP.…”

“…Changes in size and composition of the intracellular CoA pool in aerobic and facultatively anaerobic bacteria Intracellular levels of three CoA molecular species in a variety of aerobic and facultatively anaerobic bacteria were analyzed using the acyl-CoA cycling method 12,15,16,23,65,84,86) . It has been demonstrated that there is a remarkable difference in the size and composition of intracellular pools of CoASH and CoA thioesters between aerobic bacteria and facultatively anaerobic bacteria.…”