Mecillinam resistance in Escherichia coli is conferred by loss of a second activity of the AroK protein

Abstract

Mecillinam, a ␤-lactam antibiotic specific to penicillin-binding protein 2 (PBP 2) in Escherichia coli, blocks cell wall elongation and, indirectly, cell division, but its lethality can be overcome by increased levels of ppGpp, the nucleotide effector of the stringent response. We have subjected an E. coli K-12 strain to random insertional mutagenesis with a mini-Tn10 element. One insertion, which was found to confer resistance to mecillinam in relA ؉ and relA strains, was mapped at 75.5 min on the E. coli map and was located between the promoters and the coding sequence of the aroK gene, which codes for shikimate kinase I, one of two E. coli shikimate kinases, both of which are involved in aromatic amino acid biosynthesis. The mecillinam resistance conferred by the insertion was abolished in a ⌬relA ⌬spoT strain completely lacking ppGpp, and it thus depends on the presence of ppGpp. Furthermore, the insertion increased the ppGpp pool approximately twofold in a relA ؉ strain. However, this increase was not observed in relA strains, although the insertion still conferred mecillinam resistance in these backgrounds, showing that mecillinam resistance is not due to an increased ppGpp pool. The resistance was also abolished in an ftsZ84(Ts) strain under semipermissive conditions, and the aroK::mini-Tn10 allele partially suppressed ftsZ84(Ts); however, it did not increase the concentration of the FtsZ cell division protein. The insertion greatly decreased or abolished the shikimate kinase activity of AroK in vivo and in vitro. The two shikimate kinases of E. coli are not equivalent; the loss of AroK confers mecillinam resistance, whereas the loss of AroL does not. Furthermore, the ability of the aroK mutation to confer mecillinam resistance is shown to be independent of polar effects on operon expression and of effects on the availability of aromatic amino acids or shikimic acid. Instead, we conclude that the AroK protein has a second activity, possibly related to cell division regulation, which confers mecillinam sensitivity. We were able to separate the AroK activities mutationally with an aroK mutant allele lacking shikimate kinase activity but still able to confer mecillinam sensitivity.

Role of 2-Phosphoglycolate Phosphatase ofEscherichia coliin Metabolism of the 2-Phosphoglycolate Formed in DNA Repair

“…Up to now, some of the gene products have been related to DNA function. For instance, the aroK gene product could have a second activity possibly related to cell division (36); methylation mediated by dam is known to be involved in several processes, such as DNA replication, gene expression and mismatch repair, among others; and the epimerase encoded by the rpe gene has been reported to be involved not only in the nonoxidative branch of the pentose phosphate pathway but also in the control of chromosome replication (28). In this context, it is tempting to speculate that the physiological role proposed here for the gph gene product matches rather well the role in DNA-related processes suggested for most of the genes in the dam-containing operon, giving further support to the function proposed for the gph gene product.…”

Metabolism Shapes the Cell

“…In E. coli, overexpression of the cell division proteins FtsQ, FtsA, and FtsZ (but not FtsZ alone) or artificially induced synthesis of (p)ppGpp bypasses the lethality associated with amdinocillin treatment, so it has been proposed that increased (p)ppGpp levels result in an enhanced cell division capacity that allows the wider cells to divide (28,29). However, subsequent studies demonstrated that increased FtsZ levels are not responsible for amdinocillin resistance, at least for the mutants that have been examined (30,31), indicating that another mechanism confers resistance.…”

“…aroK encodes a shikimate kinase that catalyzes the ATP-dependent conversion of shikimate to shikimate-3-phosphate in the aromatic amino acid synthesis pathway (31). The mutant also shows partial rescue of the filamentation and lethality effects associated with ftsZ84(Ts), a temperature-sensitive allele of ftsZ, indicating that aroK inactivation acts as a general suppressor of defects in cell envelope synthesis.…”

“…The mutant also shows partial rescue of the filamentation and lethality effects associated with ftsZ84(Ts), a temperature-sensitive allele of ftsZ, indicating that aroK inactivation acts as a general suppressor of defects in cell envelope synthesis. Based on the fact that FtsZ levels were not detectably different in the aroK ftsZ84 double mutant strain and that the amdinocillin resistance was not dependent on AroK's characterized enzymatic activity or relA, the authors concluded that AroK likely possesses a second, moonlighting function related to cell division regulation (31). Another possibility is that aroK inactivation causes a change in carbon flux.…”

Expression of Rat Sperm Flagellum-Movement Associated Protein Genes under 2,3,7,8-Tetrachlorodibenzo-p-dioxin Treatment

“…17) Because these proteins appear in prokaryotes, their physiological importance in sperm cells is not certain. AK activity is necessary for ATP synthesis and to maintain an adequate concentration of ATP in eukaryotic cells.…”