Dataset Information

Identification of a novel UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from Vibrio fischeri that confers high fosfomycin resistance in Escherichia coli.

ABSTRACT: MurA [UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase] is a key enzyme involved in bacterial cell wall peptidoglycan synthesis and a target for the antimicrobial agent fosfomycin, a structural analog of the MurA substrate phosphoenol pyruvate. In this study, we identified, cloned and sequenced a novel murA gene from an environmental isolate of Vibrio fischeri that is naturally resistant to fosfomycin. The fosfomycin resistance gene was isolated from a genomic DNA library of V. fischeri. An antimicrobial agent hypersensitive strain of Escherichia coli harboring murA from V. fischeri exhibited a high fosfomycin resistance phenotype, with minimum inhibitory concentration of 3,000 microg/ml. The cloned murA gene was 1,269 bp long encoding a 422 amino acid polypeptide with an estimated pI of 5.0. The deduced amino acid sequence of the putative protein was identified as UDP-NAG enolpyruvyl transferase by homology comparison. The MurA protein with an estimated molecular weight of 44.7 kDa was expressed in E. coli and purified by affinity chromatography. MurA of V. fischeri will be a useful target to identify potential inhibitors of fosfomycin resistance in pharmacological studies.

SUBMITTER: Kumar S

PROVIDER: S-EPMC2673340 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Publications

Identification of a novel UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from Vibrio fischeri that confers high fosfomycin resistance in Escherichia coli.

Kumar Sanath S Parvathi Ammini A Hernandez Ricardo L RL Cadle Kathleen M KM Varela Manuel F MF

Archives of microbiology 20090311 5

MurA [UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase] is a key enzyme involved in bacterial cell wall peptidoglycan synthesis and a target for the antimicrobial agent fosfomycin, a structural analog of the MurA substrate phosphoenol pyruvate. In this study, we identified, cloned and sequenced a novel murA gene from an environmental isolate of Vibrio fischeri that is naturally resistant to fosfomycin. The fosfomycin resistance gene was isolated from a genomic DNA library of V. fischeri ...[more]

PMID: 19277606

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Project description:Organisms of Chlamydia spp. are obligate intracellular, gram-negative bacteria with a dimorphic developmental cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial anomaly refers to the fact that cell wall-active antibiotics inhibit Chlamydia growth and peptidoglycan (PG) synthesis genes are present in the genome, yet there is no biochemical evidence for synthesis of PG. In this work, we undertook a genetics-based approach to reevaluate the chlamydial anomaly by characterizing MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase that catalyzes the first committed step of PG synthesis. The murA gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible, glucose-repressible ara promoter and transformed into Escherichia coli. After transduction of a lethal DeltamurA mutation into the strain, viability of the E. coli strain became dependent upon expression of the C. trachomatis murA. DNA sequence analysis of murA from C. trachomatis predicted a cysteine-to-aspartate change in a key residue within the active site of MurA. In E. coli, the same mutation has previously been shown to cause resistance to fosfomycin, a potent antibiotic that specifically targets MurA. In vitro activity of the chlamydial MurA was resistant to high levels of fosfomycin. Growth of C. trachomatis was also resistant to fosfomycin. Moreover, fosfomycin resistance was imparted to the E. coli strain expressing the chlamydial murA. Conversion of C. trachomatis elementary bodies to reticulate bodies and cell division are correlated with expression of murA mRNA. mRNA from murB, the second enzymatic reaction in the PG pathway, was also detected during C. trachomatis infection. Our findings, as well as work from other groups, suggest that a functional PG pathway exists in Chlamydia spp. We propose that chlamydial PG is essential for progression through the developmental cycle as well as for cell division. Elucidating the existence of PG in Chlamydia spp. is of significance for the development of novel antibiotics targeting the chlamydial cell wall.

Project description:UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosamine-1-phosphate transferase (GPT) is the first enzyme in the dolichol pathway of protein N-glycosylation, and is implicated in the developmental programmes of a variety of eukaryotes. In the present study we describe the effects of all-trans-retinoic acid (RA) on the levels of GPT protein and enzymic activity, and on the transcription rate of the GPT gene, in mouse P19 teratocarcinoma cells. RA caused a dose-dependent and protein-synthesis-dependent induction of enzyme activity. The maximum induction of GPT activity (about 3-fold) required 2 days of exposure to 1 microM RA. Induced GPT activity also resulted in an increase in the rate of incorporation of [3H]mannose into Glc3Man9GlcNAc2. Enzymic activities paralleled GPT gene expression. The GPT gene was induced (2-fold) after 7 h of RA treatment. An approx. 3-fold increase in a 48 kDa GPT protein and approx. 4-fold increases in the levels of three GPT transcripts (1.8, 2.0 and 2.2 kb) were observed after 2 days of RA treatment. The enhanced levels of GPT protein and mRNAs began to decline 3 days after the initiation of differentiation, and GPT expression was down-regulated during cellular differentiation. GPT activity decreased about 2. 8-fold to a constant level in differentiated P19 cells. The results indicate that the RA-induced enzyme activity was mainly determined by increased transcription of the GPT gene. RA-treated P19 cells were about 4-fold more resistant to tunicamycin, a fungal antibiotic which inhibits GPT, than were control cells. In addition, GPT activity in membranes from RA-treated P19 cells exhibited approx. 4-fold increased resistance to tunicamycin compared with activity in membranes from untreated control cells, demonstrating that resistance to tunicamycin is correlated with induced GPT activity. Furthermore, increased GPT activity had regulatory significance with regard to the rate of incorporation of [3H]mannose into Glc3Man9GlcNAc2-P-P-dolichol and into glycoproteins. Together, the data provide additional insights into the hormonal regulation of GPT and present evidence that the RA-mediated induction of GPT has a regulatory impact on the dolichol pathway.

Dataset Information

Identification of a novel UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from Vibrio fischeri that confers high fosfomycin resistance in Escherichia coli.

Publications

Identification of a novel UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from Vibrio fischeri that confers high fosfomycin resistance in Escherichia coli.

Similar Datasets

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