Quantification of polyprenyl diphosphates in Escherichia coli cells using high-performance liquid chromatography.

Dephosphorylation of undecaprenyl diphosphate is a crucial step in the synthesis of undecaprenyl phosphate, which is essential for cell wall synthesis. We have developed a method for the quantification of intracellular polyprenyl diphosphates, which have never before been measured directly. Polyprenyl phosphates and diphosphates prepared by chemical phosphorylation of polyprenols from Staphylococcus aureus were used to establish the conditions for fractionation by ion-exchange chromatography and high-performance liquid chromatography (HPLC). By using an elution solvent containing tetraethylammonium phosphate as an ion-pair reagent for HPLC, polyprenyl phosphate and polyprenyl diphosphate with carbon numbers from 40 to 55 could be detected as separate peaks from the reversed-phase column. This analytical method was applied to lipids extracted from Escherichia coli to determine the intracellular levels of octaprenyl phosphate, undecaprenyl phosphate, octaprenyl diphosphate, and undecaprenyl diphosphate. This is the first report of separate measurement of cellular levels of polyprenyl phosphates and polyprenyl diphosphates.

Population Analysis of Daptomycin-non-Susceptible Methicillin-Resistant Staphylococcus aureus Reveals the Presence of Variants That Contribute to Daptomycin Resistance.

In this study, population analysis (PA) of methicillin-resistant Staphylococcus aureus (MRSA), before and after long-duration daptomycin (DAP) treatment, was used to detect subpopulations with different susceptibilities to DAP and to verify the changes in the number of resistant cells. Furthermore, we aimed to characterize the bacteriology of the variants present in the non-susceptible cell subpopulation. A DAP non-susceptible (NS) MRSA phenotype (D2) that emerged from a DAP- susceptible MRSA phenotype (D1) during treatment of an open wound, was used for testing. We performed bacteriological and genetic analyses of cryptic DAP-NS MRSA variants detected by PA to study the variants present in the resistant cell subpopulation. PA results suggest that MRSA adapted to survival in the presence of DAP are selected leading to reduced susceptibility. Within the cell population growing in media containing 2.0 mg/L of DAP, three variants with different pigment production and colony size were detected. Variant 3 was an orange colony due to enhanced production of staphyloxanthin. Our results revealed that the DAP minimum inhibitory concentration (MIC) value increased two-fold (4 mg/L) in variant 3, in which pigment production was most enhanced, compared to the parental strain D2. In conclusion, our results indicate that long-duration DAP treatment can lead to the emergence and increased proportion of DAP-NS subpopulations. Furthermore, slow-growing variants that can be detected only under antimicrobial selective pressure are present among DAP-NS cells, suggesting that these variants may also contribute to the development of DAP resistance.

Liquid chromatography-mass spectrometry analyses of polyprenyl phosphates in Escherichia coli cells in which genes for isoprenoid synthesis are amplified.

Overproduction of isopentenyl diphosphate by the amplification of the genes for the methylerythritol 4-phosphate pathway, dxs and dxr, is known to be deleterious for the growth of Escherichia coli. We hypothesized that overproduction of one of the endogenous isoprenoids, in addition to isopentenyl diphosphate itself, might be the cause of the reported reduced growth rate and attempted to identify the causative agent. In order to analyze polyprenyl phosphates, they were methylated by the reaction with diazomethane. The resulting dimethyl esters of polyprenyl phosphates with carbon numbers from 40 to 60 were quantitated by high-performance liquid chromatography-mass spectrometric analysis detecting ion peaks of the sodium ion adducts. The E. coli was transformed by a multi-copy plasmid carrying both the dxs and dxr genes. Amplification of dxs and dxr significantly increased the levels of polyprenyl phosphates and 2-octaprenylphenol. The levels of Z,E-mixed polyprenyl phosphates with carbon numbers of 50-60 in the strain in which ispB was co-amplified with dxs and dxr were lower than those in the control strain where only dxs and dxr were amplified. The levels of (all-E)-octaprenyl phosphate and 2-octaprenylphenol in the strains in which ispU/rth or crtE was co-amplified with dxs and dxr were lower than those in the control strain. Although the increase in the level of each isoprenoid intermediate was blocked, the growth rates of these strains were not restored. Neither polyprenyl phosphates nor 2-octaprenylphenol can be determined to be the cause of the growth rate reduction seen with dxs and dxr amplification.