The Escherichia coli mutant requiring D-glutamic acid is the result of mutations in two distinct genetic loci

Abstract

D-Glutamic acid is an essential component of bacterial cell wall peptidoglycan in both gram-positive and gram-negative bacteria. Very little is known concerning the genetics and biochemistry of D-glutamate production in most bacteria, including Escherichia coli. Evidence is presented in this report for the roles of two distinct genes in E. coli WM335, a strain which is auxotrophic for D-glutamate. The first gene, which restores D-glutamate independence in WM335, was mapped, cloned, and sequenced. This gene, designated dga, is a previously reported open reading frame, located at 89.8 min on the E. coli map. The second gene, giS, is located at 82 min. gitS encodes a protein that is involved in the transport of D-and L-glutamic acid into E. cofl, and the gitS gene of WM335 was found to contain two missense mutations. To construct D-glutamate auxotrophs, it is necessary to transfer sequentially the mutated gitS locus, and then the mutated dga locus into the recipient. The sequences of the mutant forms of both dga and gitS are also presented.One of the unique aspects of the bacterial cell wall is the presence of D-amino acids in the peptidoglycan. The D-amino acids confer several essential properties on the peptidoglycan, including conformation and resistance to proteases. For the D-alanine moiety, the pathway for biosynthesis is well characterized (3, 33). Likewise, the mesodiaminopimelic acid biosynthetic scheme and its relation to lysine biosynthesis is known (4). However, understanding of the route of D-glutamic acid biosynthesis has been relatively sparse, with very little information available concerning D-glutamate in enteric bacteria, such as Escherichia coli.Early studies investigated D-glutamate biosynthesis in either Bacillus sp. and in lactobacilli and pediococci. In Bacillus sp., D-glutamate is formed by a D-amino acid transaminase utilizing D-alanine and a-ketoglutarate (15,(29)(30)(31). In contrast, Lactobacillus sp. and Pediococcus sp. apparently use a racemase that converts L-glutamate to D-glutamate (21, 22). Thus, at least two different biosynthetic routes for D-glutamate are known in bacteria. In E. coli, the overall glutamic acid pool is high, and approximately 10% of the pool is in the form of D-glutamic acid (18). Nevertheless, attempts to define the enzymatic mechanism for D-glutamate synthesis in E. co0i have all met with failure to date (8,21,22,29). In an attempt to clarify the situation in E. coli, we initiated investigation of the genes involved in D-glutamate biosynthesis. Only one D-glutamate auxotrophic mutant in E. coli has been reported to date. The mutation in E. coli B/r WM335, which renders the bacteria absolutely dependent on an exogeneous source of D-glutamate, has been mapped to the 70-to 90-min region on the E. coli chromosome (17). In the present study, we refine the map position to 89.8 min. The mutation was found to reside in a previously reported unidentified open reading frame (2). Upon further investigation, it was found that the D-glutamic acid mutant strain E. coil B/r...

Investigation of the Essentiality of Glutamate Racemase in Mycobacterium smegmatis

“…Since D-glutamate is known to be poorly transported across bacterial membranes (24, 25), we concluded that larger amounts of D-glutamate supplementation likely were required for true ⌬murI mutants to survive. This conclusion is consistent with studies in E. coli showing that a ⌬murI strain could not be obtained until a mutant strain (WM335) was isolated that contained a mutation allowing for increased transport of Dglutamate across the plasma membrane (24,26).…”

Characteristics of a New Enantioselective Thermostable Dipeptidase from Brevibacillus borstelensis BCS-1 and Its Application to Synthesis of a d -Amino-Acid-Containing Dipeptide

“…B. borstelensis BCS-1 was cultured at 55°C in Luria-Bertani (LB) medium. E. coli strain WM335 (kindly donated by Walter Messer of the Max-Planck Institute of Molecular Genetics, Berlin, Germany), which requires D-Glu for growth, was cultured at 37°C and used as the cloning host strain (10). E. coli XL1-Blue (Stratagene, La Jolla, Calif.) was used as the expression host strain.…”

Directed Evolution of Ionizing Radiation Resistance in Escherichia coli

“…The ClpP and ClpX proteins are components of a proteolytic system responsible for the turnover of at least 60 E. coli proteins, including chaperones, proteins involved in energy production, transcriptional regulators, and others (3,18). GltS is a membrane-bound glutamate transporter (15,48). BglH is an outer membrane porin for ␤-glucosides (2).…”