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.

Does sub-culturing of positive MRSA blood cultures affect vancomycin MICs?

Introduction. Empirical vancomycin (VAN) treatment failure for methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia, with significantly higher mortality, has been reported for MRSA strains with reduced VAN susceptibility.Aim. Our goal was to study the effect of sub-culture on VAN minimum inhibitory concentration (MIC) values compared to direct susceptibility of MRSA-positive blood cultures.Methodology. Using 19 MRSA-positive blood cultures and 19 seeded MRSA-positive blood cultures, we compared the VAN MICs from direct susceptibility testing of MRSA-positive blood cultures and MRSA sub-cultured from positive blood cultures.Results. In comparing direct VAN MICs from MRSA-positive blood cultures and standard agar dilution, nearly half of the MICs from agar dilution were lower, with one sample decreasing from 1.5 to 0.75 µg ml-1. Furthermore, in seeded blood cultures, 80 % or more showed lower values from standard agar dilution compared to direct VAN MICs.Conclusion. Our results reveal a trend towards lower MICs after positive blood culture isolates are sub-cultured. Some clinical failures among MRSA infections treated with VAN may result from this phenomenon.

Characterization of compensatory mutations associated with restoration of daptomycin-susceptibility in daptomycin non-susceptible methicillin-resistant Staphylococcus aureus and the role mprF mutations.

The objective of this study was to investigate the underlying mechanism explaining reversion of clinical DAP non-susceptible (NS) MRSA isolates to DAP-susceptible (S) by analysis of genomic and cell wall characteristics of clinical DAP-NS MRSA and DAP-S MRSA isolates as well as in vitro revertant DAP-S MRSA using whole genome sequencing (WGS) and analysis of biological properties. WGS of the 4 clinical DAP-NS MRSA revealed mprF mutations resulting in amino acid substitutions or deletion. These same amino acid substitutions and deletion were also observed in the 4 in vitro revertant DAP-S strains. While WGS identified the presence of the same mprF mutations in both the DAP-NS and in vitro DAP-S revertant strains, new mutations were also detected in other genes and intergenic regions of in vitro DAP-S revertant strains. Transmission electron microscopy to assess cell-wall (CW) thickness of 4 sets strains (pre- and post-DAP therapy isolates and in vitro DAP-S revertant) showed that 3 of the 4 isolates developed increased thickness of the CW after DAP therapy. After reversion to DAP susceptibility, CW thickness was decreased to the same level as DAP-S MRSA. Our results indicate that in vitro conversion of DAP-NS MRSA to DAP-S is independent of mprF gene mutations and may be partially explained by a change in CW thickness. However, as some strains showed no change in the CW, further studies are required to elucidate the different mechanisms of resistance to DAP, and factors for conversion of DAP-NS to DAP-S.

Suppression of phenotype of Escherichia coli mutant defective in farnesyl diphosphate synthase by overexpression of gene for octaprenyl diphosphate synthase.

We investigated suppression of the slow growth of an Escherichia coli ispA null mutant lacking farnesyl diphosphate (FPP) synthase (i.e. IspA) by plasmids carrying prenyl diphosphate synthase genes. The growth rates of ispA mutant-transformants harboring a medium-copy number plasmid that carries ispA or ispB were almost the same as that of the wild-type strain. Although the level of FPP in the transformant with the ispA plasmid was almost the same as that in the wild-type strain, the level in the transformant with the ispB plasmid was as low as that in the ispA mutant. Purified octaprenyl diphosphate synthase (IspB) could condense isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP) to form octaprenyl diphosphate and nonaprenyl diphosphate. It is possible that suppression of the slow growth of the ispA mutant by ispB was due to condensation of IPP not only with FPP but also with DMAPP by octaprenyl diphosphate synthase.

Undecaprenyl phosphate metabolism in Gram-negative and Gram-positive bacteria.

Undecaprenyl phosphate (UP) is essential for the biosynthesis of bacterial extracellular polysaccharides. UP is produced by the dephosphorylation of undecaprenyl diphosphate (UPP) via de novo synthetic and recycling pathways. Gram-positive bacteria contain remarkable amounts of undecaprenol (UOH), which is phosphorylated to UP, although UOH has not been found in Gram-negative bacteria. Here, current knowledge about UPP phosphatase and UOH kinase is reviewed. Dephosphorylation of UPP is catalyzed by a BacA homologue and a type-2 phosphatidic acid phosphatase (PAP2) homologue. The presence of one of these UPP phosphatases is essential for bacterial growth. The catalytic center of both types of enzyme is located outside the cytoplasmic membrane. In Gram-positive bacteria, an enzyme homologous to DgkA, which is the diacylglycerol kinase of Escherichia coli, catalyzes UOH phosphorylation. The possible role of UOH and the significance of systematic construction of Staphylococcus aureus mutants to determine UP metabolism are discussed.

Molecular Characterization of Fluoroquinolone-Resistant Moraxella catarrhalis Variants Generated In Vitro by Stepwise Selection.

Moraxella catarrhalis causes respiratory infections. In this study, fluoroquinolone-resistant strains were selected in vitro to evaluate the mechanism of fluoroquinolone resistance. Strains with reduced fluoroquinolone susceptibility were obtained by stepwise selection in levofloxacin, and fluoroquinolone targets gyr and par were sequenced. Six novel mutations in GyrA (D84Y, T594dup, and A722dup), GyrB (E479K and D439N), and ParE (Q395R) involved in M. catarrhalis resistance to fluoroquinolones were revealed.

Non-invasive measurement of melanin-derived radicals in living mouse tail using X-band EPR.

The aim of this experiment is to measure in vivo generation of melanin-derived radicals non-invasively, as a quantifiable index of radio-biological effect. Melanin-derived radicals in a living intact mouse tail tip were non-invasively measured in very simple way using an X-band electron paramagnetic resonance spectrometer. Colored mouse strains, C57BL/6NCr, BDF1, and C3H/He, have clear EPR signal corresponding to melanin-derived radicals in the tail tip; however, albino mouse strains, BALB/cCr, ddY, ICR, have no EPR signals. An X-ray fraction of 2 Gy/day (1 Gy/min) was repeatedly irradiated to a C3H/He mouse tail skin every Monday to Friday for 4 weeks. In comparison to before starting irradiation, the C3H/He mouse tail skin became darker, like a suntan. The melanin-derived radicals in C3H/He mouse tail skin were increased in association with X-ray fractions. Melanin-derived radicals in mouse tail skin can be readily and chronologically measurable by using X-band EPR spectrometer, and can be a marker for a radiobiological effect in the skin.

[Decreasing Susceptibility of MRSA to Daptomycin During the Course of Treatment].

Methicillin-resistant Staphylococcus aureus (MRSA) with decreased susceptibility to daptomycin (DAP) were isolated from 4 patients receiving DAP from November 2013 to May 2014. These patients were treated with DAP for more than 7 days in all the cases. The pulsed-field gel electrophoresis (PFGE) patterns for MRSA isolates recovered from each patient pre- and post-DAP therapy were identical. Sequencing of mprF detected 2 amino acid substitutions (T345I or L826F) in 2 of the isolates. These results suggest that in vivo MRSA was resistant to DAP during DAP therapy. Furthermore, the MICs for DAP can vary by±1 dilution depending on the susceptibility test. When testing DAP susceptibility, there is a need to monitor reproducibility using different susceptibility tests, including the CLSI method.

Accumulation of alkaline earth metals by the green macroalga Bryopsis maxima.

Twenty-five days after the disaster at the Fukushima Daiichi nuclear power plant in 2011, we collected samples of the green macroalga Bryopsis maxima from the Pacific coast of Japan. Bryopsis maxima is a unicellular, multinuclear, siphonous green macroalga. Radiation analysis revealed that B. maxima emitted remarkably high gamma radiation of (131)I, (134)Cs, (137)Cs, and (140)Ba as fission products of (235)U. Interestingly, B. maxima contained naturally occurring radionuclides derived from (226)Ra and (228)Ra. Analysis of element content revealed that B. maxima accumulates many ocean elements, especially high quantities of the alkaline earth metals Sr (15.9 g per dry-kg) and Ba (3.79 g per dry-kg), whereas Ca content (12.5 g per dry-kg) was lower than that of Sr and only 61 % of the mean content of 70 Japanese seaweed species. Time-course analysis determined the rate of radioactive (85)Sr incorporation into thalli to be approximately 0.13 g Sr per dry-kg of thallus per day. Subcellular fractionation of B. maxima cells showed that most of the (85)Sr was localized in the soluble fraction, predominantly in the vacuole or cytosol. Given that (85)Sr radioactivity was permeable through a dialysis membrane, the (85)Sr was considered to be a form of inorganic ion and/or bound with a small molecule. Precipitation analysis with sodium sulfate showed that more than 70% of the Sr did not precipitate as SrSO4, indicating that a proportion of the Sr may bind with small molecules in B. maxima.

Identification of tomoregulin-1 as a novel addicsin-associated factor.

Addicsin is a novel factor encoding a 23-kDa hydrophobic protein that is highly upregulated in the amygdala nuclei of morphine-administered mice. It is a murine homolog of human JWA and rat glutamate transporter-associated protein 3-18 (GTRAP3-18), a negative modulator of the neural glutamate transporter excitatory amino acid carrier 1 (EAAC1). Recent findings demonstrated that addicsin participates in various physiological processes by forming hetero- or homomultimeric complexes. However, the binding targets and molecular functions of addicsin remain largely unknown. To identify potential factors that associate with mouse addicsin, we performed a yeast two-hybrid screen using a 17-day-old mouse whole embryo cDNA library. We identified tomoregulin-1 (TR1) as a novel addicsin-associated factor. TR1, a type I transmembrane protein containing two follistatin-like modules and an epidermal growth factor-like domain, participates in nodal and bone morphogenetic protein signaling. Immunoprecipitation assays demonstrated that TR1 bound to addicsin, and that amino acids 145-188 of addicsin and amino acids 228-266 of TR1 were important for the formation of the addicsin-TR1 heterocomplex. The double-fluorescent immunohistochemical analysis revealed that addicsin and TR1 were coexpressed in neurons in the mature mouse brain regions tested. Moreover, TR1 showed a punctuate pattern throughout the cell, with preferential expression on the cell surface when expressed alone. However, TR1 predominantly redistributed to the endoplasmic reticulum (ER) when coexpressed with addicsin. Furthermore, coexpression of an addicsin mutant that lacked TR1 binding ability had little effect on the distribution of TR1. Biotinylation assays showed that coexpression of addicsin with TR1 suppressed the cell surface expression of TR1. Wound-healing assays demonstrated that the interaction of addicsin with TR1 had a significant effect on cell migration. These findings demonstrate that addicsin in the ER controls intracellular TR1 trafficking from the ER to plasma membrane and regulates cell migration through its interaction with TR1.

Involvement of an essential gene, mviN, in murein synthesis in Escherichia coli.

We isolated a temperature-sensitive mutant with a mutation in mviN, an essential gene in Escherichia coli. At the nonpermissive temperature, mviN mutant cells swelled and burst. An intermediate in murein synthesis, polyprenyl diphosphate-N-acetylmuramic acid-(pentapeptide)-N-acetyl-glucosamine, accumulated in mutant cells. These results indicated that MviN is involved in murein synthesis.

Short-chain prenyl diphosphate synthase that condenses isopentenyl diphosphate with dimethylallyl diphosphate in ispA null Escherichia coli strain lacking farnesyl diphosphate synthase.

A short-chain prenyl diphosphate synthase in an Escherichia coli mutant that lacked the gene coding for farnesyl diphosphate synthase, ispA, was separated from other prenyl diphosphate synthases by DEAE-Toyopearl column chromatography. The purified enzyme catalyzed the condensation of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl diphosphate and geranylgeranyl diphosphate.

Analysis of six new genes encoding lysis proteins and coat proteins in Escherichia coli group A RNA phages.

Group A RNA phages consist of four genes-maturation protein, coat protein, lysis protein and replicase genes. We analyzed six plasmids containing lysis protein genes and coat protein genes of Escherichia coli group A RNA phages and compared their amino acid sequences with the known proteins of E. coli(group A), Pseudomonas aeruginosa(PP7) RNA phages and Rg-lysis protein from Qbeta phage. The size of lysis proteins was different by the groups but the coat proteins were almost the same size among phages. The phylogenetic analysis shows that the sub-groups A-I and A-II of E. coli RNA phages were clearly dispersed into two clusters.

Disruption of the structural gene for farnesyl diphosphate synthase in Escherichia coli.

The chromosomal ispA gene encoding farnesyl diphosphate synthase of Escherichia coli was disrupted by inserting a neo gene cassette. The null ispA mutants were viable. The growth yield of the mutants was 70% to 80% of that of the wild-type strain under aerobic conditions, and was almost the same as the wild-type under anaerobic conditions. The levels of ubiquinone-8 and menaquinone-8 were both significantly lower (less than 13% and 18% of normal, respectively) in the mutants than in the wild-type. The undecaprenyl phosphate level in the mutants was modestly lower (40% to 70% of normal) than in the wild-type strain. Thus the synthesis of all-E-octaprenyl diphosphate, the precursor of ubiquinone-8 and menaquinone-8, was decreased more severely than that of Z,E-mixed undecaprenyl diphosphate, the precursor of undecaprenyl monophosphates, under the conditions where the synthesis of farnesyl diphosphate was decreased. The condensation of isopentenyl diphosphate with dimethylallyl diphosphate was detected in the cell-free extracts of the mutants, although it was 5% of that in the wild-type strain. A low level of farnesyl diphosphate seems to be synthesized in the mutants by other prenyltransferases such as octaprenyl diphosphate synthase or undecaprenyl diphosphate synthase.

Interaction of the Escherichia coli lipoprotein NlpI with periplasmic Prc (Tsp) protease.

Escherichia coli spr (suppressor of prc) mutants and nlpI mutants show thermosensitive growth. The thermosensitivity of the spr mutants was suppressed by the nlpI mutations. Expression of the fusion genes encoding hexa-histidine-tagged NlpI (NlpI-His) and purification of the tagged NlpI showed that NlpI-His bound with Prc protease and IbpB chaperone. NlpI-His with the amino acid substitution of G103D did not bind with either of these proteins, while NlpI-His variants (NlpI-284-His, NlpI-Q283-His, and NlpI-G282-His) lacking 10 to 12 residues from the carboxy terminus bound with both proteins. The tagged NlpI lacking 11 amino acid residues from the carboxy terminus was processed by Prc, but that lacking 12 residues was not. The thermosensitivity of the nlpI mutant was corrected by the production of the former NlpI variant, but not by production of the latter. Expression of the truncated NlpI that lacked 10 or 11 residues from the carboxy terminus corrected the thermosensitivity of the prc nlpI double mutant, while expression of the full-length NlpI did not. Thus, it was suggested that NlpI was activated by Prc protease processing.

Molecular cloning and heterologous expression of the C-13 phenylpropanoid side chain-CoA acyltransferase that functions in Taxol biosynthesis.

The structural pharmacophore of Taxol, responsible for binding the N terminus of the beta-subunit of tubulin to arrest cell proliferation, comprises, in part, the 13-O-(N-benzoyl-3-phenylisoserinoyl) side chain. To identify the side chain transferase of Taxol biosynthesis, a set of transacylases obtained from an enriched cDNA library (constructed from mRNA isolated from Taxus cuspidata cells induced with methyl jasmonate for Taxol production) was screened. A cDNA clone (designated TAX7) encoding a taxoid C-13 O-phenylpropanoyltransferase was isolated which yielded a recombinant enzyme that catalyzes the selective 13-O-acylation of baccatin III with beta-phenylalanoyl CoA as the acyl donor to form N-debenzoyl-2'-deoxytaxol. This enzymatic product was converted to 2'-deoxytaxol by chemical N-benzoylation, and the identity of this derivative was confirmed by spectrometric analyses. The full-length cDNA has an ORF of 1,335 bases and encodes a 445-aa protein with a calculated molecular weight of 50,546. Evaluation of kinetic parameters revealed K(m) values of 2.4 +/- 0.5 microM and 4.9 +/- 0.3 microM for baccatin III and beta-phenylalanoyl-CoA, respectively. The pH optimum for the recombinant O-(3-amino-3-phenylpropanoyl)transferase is at 6.8. Identification of this clone completes acquisition of the five aroyl/acyltransferases involved in the biosynthesis of Taxol. Application of these transacylase genes in suitable host cells can improve the production yields of Taxol and could enable the preparation of second-generation Taxol analogs possessing greater bioactivity and improved water solubility.