The murI gene of Escherichia coli is an essential gene that encodes a glutamate racemase activity

Doublet, Patricia; Heijenoort, Jean van; Bohin, Jean-Pierre; Mengin‐Lecreulx, Dominique

doi:10.1128/jb.175.10.2970-2979.1993

Cited by 95 publications

(82 citation statements)

References 43 publications

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“…The dga (murl) gene was identified by its ability to complement the D-glutamate auxotrophic phenotype of the only known Dglutamic-acid-requiring mutant, E. coli B/r WM335 (9,12), and could be stably maintained on high-copy-number pUC vectors (5,6). Doublet et al recently reported data that suggest that dga (murI) encodes a racemase enzyme in E. coli (4). This finding is supported by our own experimental data and protein sequence analysis.…”

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confidence: 74%

The Escherichia coli Dga (MurI) protein shares biological activity and structural domains with the Pediococcus pentosaceus glutamate racemase

et al. 1994

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The Pediococcus pentosaceus glutamate racemase gene product complemented the D-glutamate auxotrophy of Escherichia coli WM335. Amino acid sequence analysis of the two proteins revealed 28% identity, primarily in six clusters scattered throughout the sequence. Further analyses indicated secondary structure similarities between the two proteins. These data support a recent report that the dga (murl) gene product is a glutamate racemase.The peptidoglycan component of the bacterial cell wall is synthesized by a number of unique enzymes (10). Several of these enzymes either synthesize D-amino acids or add them to the monomeric subunit precursor in the cytoplasm. One of these enzymatic activities, D-glutamic acid biosynthesis, has not been characterized in most species of bacteria, including Escherichia coli. Early studies with a few species revealed two different biosynthetic routes of D-glutamic acid biosynthesis. In Bacillus spp., D-glutamate is formed from D-alanine and a-ketoglutarate by a D-amino acid transaminase (11,18,20,21), while Lactobacillus spp. (7,8,19) and Pediococcus spp. use a racemase which converts L-glutamate to D-glutamate (1, 2, 13, 14). All prior attempts to detect either of these activities in E. coli had been unsuccessful (5,13,14). In addition, the gene(s) responsible for D-glutamate biosynthesis in this organism also had not been identified until recently (5, 6). The dga (murl) gene was identified by its ability to complement the D-glutamate auxotrophic phenotype of the only known Dglutamic-acid-requiring mutant, E. coli B/r WM335 (9, 12), and could be stably maintained on high-copy-number pUC vectors (5, 6). Doublet et al. recently reported data that suggest that dga (murI) encodes a racemase enzyme in E. coli (4). This finding is supported by our own experimental data and protein sequence analysis.Glutamate racemase complements an E. coli D-glutamate auxotroph. Nakajima et al. described the cloning of the glutamate racemase gene of Pediococcus pentosaceus and its expression in E. coli (13). Plasmid pICR222, which consists of a 1.65-kb HindIII-EcoRI fragment containing the racemase gene cloned into pUC18 (Fig. 1) Fig. 1]). Subsequently, 110 bp was removed from the ORF on pRDD022 via a DraII deletion (recombinant plasmid designated pRDD023 [ Fig. 1]). Both pRDD022 and pRDD023 were transformed into WM335 and checked for their ability to complement the D-glutamic acid-requiring phenotype. Plasmid pRDD022 was able to complement the phenotype, while plasmid pRDD023 could not (data not shown). These data provide strong evidence that the 795-bp ORF encodes an activity which allows WM335 to grow normally in the absence of exogenous Dglutamic acid. Since pUC19 is present in E. coli cells in high copy numbers (>100 copies), the ORF was subcloned into a low-copy-number vector, pWSK29 (approximately five copies per cell [22]), to determine whether a relatively low gene dosage would still complement WM335. The recombinant plasmid, designated pRDD024 (Fig. 1)

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confidence: 74%

The Escherichia coli Dga (MurI) protein shares biological activity and structural domains with the Pediococcus pentosaceus glutamate racemase

et al. 1994

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“…Involvement of the gbnU gene product in peptidoglycan synthesis. Since the effects on bacterial shape and cell integrity provoked by the defect of glmU resembled the phenotypes of previously characterized mutants defective in peptidoglycan synthesis (4,8,22,23,26,33,37), the effect of a temperature upshift on peptidoglycan synthesis in both strains JM83(pGMU) and UGS83 was determined. In order to estimate the rate of synthesis of this macromolecule, we directly quantitated the cell peptidoglycan content by amino acid and amino sugar analyses of isolated sacculi.…”

Section: Resultsmentioning

confidence: 99%

“…The pGM7 plasmid (10) carrying the 4.6-kb NcoI-ClaI fragment that encompasses both the glmU and glmS genes (Fig. 2) (8).…”

Section: Resultsmentioning

confidence: 99%

Identification of the glmU gene encoding N-acetylglucosamine-1-phosphate uridyltransferase in Escherichia coli

1993

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The physiological properties of the EcoURF-1 open reading frame, which precedes the gbnS gene at 84 min on the Escherichia coli chromosome (J. E. Walker, N. J. Gay, M. Saraste, and A. N. Eberle, Biochem. J. 224:799-815, 1984), were investigated. A thermosensitive conditional mutant in which the synthesis of the gene product was impaired at 43°C was constructed. The inactivation of the gene in exponentially growing cells rapidly inhibited peptidoglycan synthesis. As a result, various alterations of cell shape were observed, and cell lysis finally occurred when the peptidoglycan content was 37% lower than that of normally growing cells. Analysis of the pools of peptidoglycan precursors revealed a large accumulation of N-acetylglucosamine-lphosphate and the concomitant depletion of the pools of the seven peptidoglycan nucleotide precursors located downstream in the pathway, a result indicating that the mutational block was in the step leading from N-acetylglucosamine-l-phosphate and UTP to the formation of UDP-N-acetylglucosamine. In vitro assays showed that the overexpression of this gene in E. col cells, directed by appropriate plasmids, led to a high overproduction (from 25-to 410-fold) of N-acetylglucosamine-l-phosphate uridyltransferase activity. This allowed us to purify this enzyme to homogeneity in only two chromatographic steps. The gene for this enzyme, which is essential for peptidoglycan and lipolysaccharide biosyntheses, was designated glnmU.

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“…We constructed an in-frame deletion of murI in our improved sacB-based suicide vector, pMP812. Since it has been shown that it is possible to rescue D-glutamate auxotrophs with exogenous D-glutamate in Bacillus subtilis and E. coli B/r and K-12 strains (Ashiuchi et al, 2007;Doublet et al, 1993;Hoffmann et al, 1972), we performed a standard two-step allelic exchange (LoVullo et al, 2006) using pMP884 (Table 1) with D-glutamic acid present in the medium. We then picked and patched 24 sucroseresistant secondary recombinants onto media with or without D-glutamic acid.…”

Section: Allelic Exchange Of Muri and Complementation With The Singlementioning

confidence: 99%

“…We chose to interrupt the production of D-glutamate by deleting the murI gene, which encodes a glutamate racemase that is essential to E. coli (Doublet et al, 1993). D-Glutamate is indispensable for the biosynthesis of peptidoglycan in most eubacteria, and is produced through two known routes: by D-amino acid transferase (D-AAT), which converts a-ketoglutarate to D-glutamate by transamination with D-alanine provided by the alanine racemase reaction; and by glutamate racemase, which produces Dglutamate through the racemization of L-glutamate (Liu et al, 1998).…”

Section: Allelic Exchange Of Muri and Complementation With The Singlementioning

confidence: 99%

Single-copy chromosomal integration systems for Francisella tularensis

et al. 2009

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Francisella tularensis is a fastidious Gram-negative bacterium responsible for the zoonotic disease tularemia. Investigation of the biology and molecular pathogenesis of F. tularensis has been limited by the difficulties in manipulating such a highly pathogenic organism and by a lack of genetic tools. However, recent advances have substantially improved the ability of researchers to genetically manipulate this organism. To expand the molecular toolbox we have developed two systems to stably integrate genetic elements in single-copy into the F. tularensis genome. The first system is based upon the ability of transposon Tn7 to insert in both a site-and orientation-specific manner at high frequency into the attTn7 site located downstream of the highly conserved glmS gene. The second system consists of a sacB-based suicide plasmid used for allelic exchange of unmarked elements with the blaB gene, encoding a b-lactamase, resulting in the replacement of blaB with the element and the loss of ampicillin resistance. To test these new tools we used them to complement a novel D-glutamate auxotroph of F. tularensis LVS, created using an improved sacB-based allelic exchange plasmid. These new systems will be helpful for the genetic manipulation of F. tularensis in studies of tularemia biology, especially where the use of multi-copy plasmids or antibiotic markers may not be suitable.

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The murI gene of Escherichia coli is an essential gene that encodes a glutamate racemase activity

Cited by 95 publications

References 43 publications

The Escherichia coli Dga (MurI) protein shares biological activity and structural domains with the Pediococcus pentosaceus glutamate racemase

The Escherichia coli Dga (MurI) protein shares biological activity and structural domains with the Pediococcus pentosaceus glutamate racemase

Identification of the glmU gene encoding N-acetylglucosamine-1-phosphate uridyltransferase in Escherichia coli

Single-copy chromosomal integration systems for Francisella tularensis

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