Identification of the Escherichia coli murI gene, which is required for the biosynthesis of D-glutamic acid, a specific component of bacterial peptidoglycan.

The murI gene of Escherichia coli, whose inactivation results in the inability to form colonies in the absence of D-glutamic acid, was identified in the 90-min region of the chromosome. The complementation of an auxotrophic E. coli B/r strain by various DNA sources allowed us to clone a 2.5-kbp EcoRI chromosomal fragment carrying the murI gene into multicopy plasmids. The murI gene corresponds to a previously sequenced open reading frame, ORF1 (J. Brosius, T. J. Dull, D. D. Sleeter, and H. F. Noller. J. Bacteriol. 148:107-127, 1987), located between the btuB gene, encoding the vitamin B12 outer membrane receptor protein, and the rrnB operon, which contains the genes for 16S, 23S, and 5S rRNAs. The murI gene product is predicted to be a protein of 289 amino acids with a molecular weight of 31,500. Attempts to identify its enzymatic activity were unsuccessful. Cells altered in the murI gene accumulate UDP-N-acetylmuramyl-L-alanine to a high level when depleted of D-glutamic acid. Pools of precursors located downstream in the pathway are consequently depleted, and cell lysis finally occurs when the peptidoglycan content is 25% lower than that of normally growing cells.     

Isolation of the murI gene from Brevibacterium lactofermentum ATCC 13869 encoding d-glutamate racemase

The murI gene encoding D-glutamate racemase plays an important role in the biosynthesis of D-glutamic acid, an essential component of cell wall peptidoglycan of almost all eubacteria. A DNA fragment that could rescue the auxotrophy of D-glutamic acid in the Escherichia coli murI mutant strain WM335 was isolated from Brevibacterium lactofermentum ATCC 13869 belonging to the coryneform bacteria. DNA sequencing reveals that it encodes a protein of 284 amino acid residues, which shows a high level of homology with D-glutamate racemases from several other bacteria.     

Staphylococcus haemolyticus contains two D-glutamic acid biosynthetic activities, a glutamate racemase and a D-amino acid transaminase.

Two D-glutamic acid biosynthetic activities, glutamate racemase and D-amino acid transaminase, have been described previously for bacteria. To date, no bacterial species has been reported to possess both activities. Genetic complementation studies using Escherichia coli WM335, a D-glutamic acid auxotroph, and cloned chromosomal DNA fragments from Staphylococcus haemolyticus revealed two distinct DNA fragments containing open reading frames which, when present, allowed growth on medium without exogenous D-glutamic acid. Amino acid sequences of the two open reading frames derived from the DNA nucleotide sequences indicated extensive identity with the amino acid sequence of Pediococcus pentosaceous glutamate racemase in one case and with that of the D-amino acid transaminase of Bacillus spp. in the second case. Enzymatic assays of lysates of E. coli WM335 strains containing either the cloned staphylococcal racemase or transminase verified the identities of these activities. Subsequent DNA hybridization experiments indicated that Staphylococcus aureus, in addition to S. haemolyticus, contained homologous chromosomal DNA for each of these genes. These data suggest that S. haemolyticus, and probably S. aureus, contains genes for two D-glutamic acid biosynthetic activities, a glutamate racemase (dga gene) and a D-amino acid transaminase (dat gene).     

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

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 (murI) gene product is a glutamate racemase.     

An Escherichia coli gene in search of a function: phenotypic effects of the gene recently identified as murI.

Earlier we reported that an open reading frame located at 89.5 min of the Escherichia coli map (ORFI) codes for a protein of unknown function that could be overexpressed and purified to homogeneity (G. Balikó, A. Raukas, I. Boros, and P. Venetianer, Mol. Gen. Genet. 211:326-331, 1988). In the work described here, we attempted to learn the function of this protein by inactivating the chromosomal gene and providing it or its deletion derivatives on temperature-sensitive plasmids. We found that the presence of the functional ORFI gene is essential; cells are not viable at the nonpermissive temperature or when the region coding for the C-terminal 50 amino acids of the protein is deleted. At intermediate temperatures or when the gene is overexpressed, characteristic changes occur in cell morphology, nucleoid separation during cell division, and supercoiling of plasmids. The possible mechanisms of these effects are discussed in view of the fact that Doublet et al. (P. Doublet, J. van Heijenoort, and D. Mengin-Lecreulx, J. Bacteriol. 174:5772-5779, 1992) recently identified the ORFI gene as murI, involved in D-glutamic acid biosynthesis.     

恶臭假单胞菌丙氨酸消旋酶基因的克隆、序列分析及表达

从恶臭假单胞菌(Pseudomonas putida)200的基因组出发,用PCR方法克隆到两个独立作用的丙氨酸消旋酶基因,称之为dadX和alr。DadX编码357个氨基酸长的多肽,计算分子量为38.82kDa,alr编码409个氨基酸长的多肽,计算分子量为44.182kDa。序列分析显示,DadX的氨基酸序列与Pseudomonas putidaKT2440,铜绿假单胞菌(Pseudomonas aeruginosa),鼠伤寒沙门氏菌(Salmonella typhimurium)和大肠杆菌(Escherichia coli)的DadX比较,相似性分别为96.64%、71.99%、44.88%和47.37%。Alr的氨基酸序列与Pseudomonas putidaKT2440比较,同源性为94.38%,而与铜绿假单胞菌(P.aeruginosa)、鼠伤寒沙门氏菌(S.typhimurium)和大肠杆菌(E.coli)的Alr比较,同源性均较低,分别为22.89%、25.72%和26.44%。在P.putida200的DadX和Alr氨基酸序列中部发现有对于酶活性至关重要的保守区域,如磷酸吡哆醛(PLP)结合位点。DadX和alr在大肠杆菌中得到表达,DadX丙氨酸消旋酶只对丙氨酸有消旋作用,而Alr丙氨酸消旋酶可以作用于丙氨酸和丝氨酸两种底物,且对丝氨酸特异性更高。Alr的表达不依赖于外源启动子,说明在其结构基因上游存在启动子结构。     

Characterization of yrpC gene product of Bacillus subtilis IFO 3336 as glutamate racemase isozyme.

Glr, the glutamate racemase of Bacillus subtilis (formerly Bacillus natto) IFO 3336 encoded by the glr gene, and YrpC, a protein encoded by the yrpC gene, which is located at a different locus from that of the glr gene in the B. subtilis genome, share a high sequence similarity. The yrpC gene complemented the D-glutamate auxotrophy of Escherichia coli WM335 cells defective in the glutamate racemase gene. Glutamate racemase activity was found in the extracts of E. coli WM335 clone cells harboring a plasmid, pYRPC1, carrying its gene. Thus, the yrpC gene encodes an isozyme of glutamate racemase of B. subtilis IFO 3336. YrpC is mostly found in an inactive inclusion body in E. coli JM109/pYRPC1 cells. YrpC was solubilized readily, but glutamate racemase activity was only slightly restored. We purified YrpC from the extracts of E. coli JM109/pYRPC2 cells using a Glutathione S-transferase Gene Fusion System to characterize it. YrpC is a monomeric protein and contains no cofactors, like Glr. Enzymological properties of YrpC, such as the substrate specificity and optimum pH, are also similar to those of Glr. The thermostability of YrpC, however, is considerably lower than that of Glr. In addition, YrpC showed higher affinity and lower catalytic efficiency for L-glutamate than Glr. This is the first example showing the occurrence and properties of a glutamate racemase isozyme.     

Cloning and characterization of the gene encoding inorganic pyrophosphatase of Escherichia coli K-12.

Escherichia coli K-12 gene ppa encoding inorganic pyrophosphatase (PPase) was cloned and sequenced. The 5' end of the ppa mRNA was identified by primer extension mapping. A typical E. coli sigma 70 promoter was identified immediately upstream of the mRNA 5' end. The structural gene of ppa contains 528 base pairs, from which a 175-amino-acid translation product, Mr 19,572, was deduced. The deduced amino acid composition perfectly fitted with that of PPase as previously determined (P. Burton, D. C. Hall, and J. Josse, J. Biol. Chem. 245:4346-4351, 1970). Furthermore, the partial amino acid sequence (residues 1 to 108) of E. coli PPase determined by S. A. Cohen (Ph.D. thesis, University of Chicago, 1978) was the same as that deduced from the nucleotide sequence. This is the first report of the cloning of a PPase gene.     

Complementation of nitrogen-regulatory (ntr-like) mutations in Rhodobacter capsulatus by an Escherichia coli gene: cloning and sequencing of the gene and characterization of the gene product.

In vivo genetic engineering by R' plasmid formation was used to isolate an Escherichia coli gene that restored the Ntr+ phenotype to Ntr- mutants of the photosynthetic bacterium Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata; J. F. Imhoff, H. G. Trüper, and N. Pfenning, Int. J. Syst. Bacteriol. 34:340-343, 1984). Nucleotide sequencing of the gene revealed no homology to the ntr genes of Klebsiella pneumoniae. Furthermore, hybridization experiments between the cloned gene and different F' plasmids indicated that the gene is located between 34 and 39 min on the E. coli genetic map and is therefore unlinked to the known ntr genes. The molecular weight of the gene product, deduced from the nucleotide sequence, was 30,563. After the gene was cloned in an expression vector, the gene product was purified. It was shown to have a pI of 5.8 and to behave as a dimer during gel filtration and on sucrose density gradients. Antibodies raised against the purified protein revealed the presence of this protein in R. capsulatus strains containing the E. coli gene, but not in other strains. Moreover, elimination of the plasmid carrying the E. coli gene from complemented strains resulted in the loss of the Ntr+ phenotype. Complementation of the R. capsulatus mutations by the E. coli gene therefore occurs in trans and results from the synthesis of a functional gene product.     

Purification and characterization of the gene 1.2 protein of bacteriophage T7

Gene 1.2 of bacteriophage T7, located near the primary origin of DNA replication at position 15.37 on the T7 chromosome, encodes a 10,059-dalton protein that is essential for growth on Escherichia coli optA1 strains (Saito, H., and Richardson, C. C. (1981) J. Virol. 37, 343-351). In the absence of the T7 1.2 and E. coli optA gene products, the degradation of E. coli DNA proceeds normally, and T7 DNA synthesis is initiated at the primary origin. However, T7 DNA synthesis ceases prematurely and the newly synthesized DNA is degraded; no viable phage particles are released. The gene 1.2 protein has been purified to apparent homogeneity from cells in which the cloned 1.2 gene is overexpressed. Purification of the [35S] methionine-labeled protein was followed by monitoring the radioactivity of the protein and by gel electrophoresis. The purified protein has been identified as the product of gene 1.2 on the basis of molecular weight and partial amino acid sequence. We have found that extracts of E. coli optA1 cells infected with T7 gene 1.2 mutants are defective in packaging exogenous T7 DNA when such extracts are prepared late in infection. Purified gene 1.2 protein restores packaging activity to these defective extracts, thus providing a biological assay for gene 1.2 protein. No specific enzymatic activity has been found associated with the purified gene 1.2 protein.     

Molecular cloning and nucleotide sequencing of the aspartate racemase gene from lactic acid bacteria Streptococcus thermophilus

The gene coding aspartate racemase (EC 5.1.1.13) was cloned from the lactic acid bacteria Streptococcus thermophilus IAM10064 and expressed efficiently in Escherichia coli. The 2.1 kilobase pairs long full length clone had an open reading frame of 729 nucleotides coding for 243 amino acids. The calculated molecular weight of 27,945 agreed well with the apparent molecular weight of 28,000 found in sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of the aspartate racemase purified from S. thermophilus. The N-terminal amino acid sequence from the purified protein exactly matches the derived sequence. In addition, the amino acid composition compiled from the derived sequence is very similar to that obtained from the purified recombinant protein. No significantly homologous proteins were found in a protein sequence data bank. Even the homology scores with alanine racemases of Salmonella typhimurium and Bacillus stearothermophilus were low. Aspartate racemase was overproduced in Escherichia coli NM522 with plasmid pAG6-2-7, which was constructed from two copies of the gene linked with a tac promoter and plasmid vector pUC18. The amount of aspartate racemase increases with the growth of E. coli and almost no degradation of the enzyme was observed. The maximum amount of the produced enzyme reached approx. 20% of the total protein of E. coli.     

Cloning of genes for bacterial glycosyltransferases. II. Selection of a hybrid plasmid carrying the rfah gene.

A hybrid ColE1 plasmid containing DNA from Escherichia coli K12 were identified which was capable of correcting the defect in UDP-galactose:lipopolysaccharide alpha1,3-galactosyltransferase in an rfaH mutant of Salmonella typhimurium. Expression of the gene for this enzyme was also demonstrated in several strains of E. coli by direct assay. The E. coli and S. typhimurium enzymes are similar in catalytic properties and immunologic specificity. The finding of the galactosyltransferase activity in E. coli extracts is surprising since the alpha1,3-galactosylglucose disaccharide which is the product of the enzyme-catalyzed reaction does not appear to be present in the E. coli lipopolysaccharide.     

UTP: alpha-D-glucose-1-phosphate uridylyltransferase of Escherichia coli: isolation and DNA sequence of the galU gene and purification of the enzyme.

The galU gene of Escherichia coli, thought to encode the enzyme UTP:alpha-D-glucose-1-phosphate uridylyltransferase, had previously been mapped to the 27-min region of the chromosome (J. A. Shapiro, J. Bacteriol. 92:518-520, 1966). By complementation of the membrane-derived oligosaccharide biosynthetic defect of strains with a galU mutation, we have now identified a plasmid containing the galU gene and have determined the nucleotide sequence of this gene. The galU gene is located immediately downstream of the hns gene, and its open reading frame would be transcribed in the direction opposite that of the hns gene (i.e., clockwise on the E. coli chromosome). The nucleotide sequences of five galU mutations were also determined. The enzyme UTP:alpha-D-glucose-1-phosphate uridylyltransferase was purified from a strain containing the galU gene on a multicopy plasmid. The amino-terminal amino acid sequence (10 residues) of the purified enzyme was identical to the predicted amino acid sequence (after the initiating methionine) of the galU-encoded open reading frame. The functional enzyme appears to be a tetramer of the galU gene product.     

The Helicobacter pylori ureC gene codes for a phosphoglucosamine mutase.

The function of UreC, the product of a 1,335-bp-long open reading frame upstream from the urease structural genes (ureAB) of Helicobacter pylori, was investigated. We present data showing that the ureC gene product is a phosphoglucosamine mutase. D. Mengin-Lecreulx and J. van Heijenoort (J. Biol. Chem. 271:32-39, 1996) observed that UreC is similar (43% identity) to the GlmM protein of Escherichia coli. Those authors showed that GlmM is a phosphoglucosamine mutase catalyzing interconversion of glucosamine-6-phosphate into glucosamine-1-phosphate, which is subsequently transformed into UDP-N-acetylglucosamine. The latter product is one of the main cytoplasmic precursors of cell wall peptidoglycan and outer membrane lipopolysaccharides. The present paper reports that, like its E. coli homolog glmM, the H. pylori ureC gene is essential for cell growth. It was known that growth of a lethal conditional glmM mutant of E. coli at a nonpermissive temperature can be restored in the presence of the ureC gene. We showed that complete complementation of the glmM mutant can be obtained with a plasmid overproducing UreC. The peptidoglycan content and the specific phosphoglucosamine mutase activity of such a complemented strain were measured; these results demonstrated that the ureC gene product functions as a phosphoglucosamine mutase. Homologs of the UreC and GlmM proteins were identified in Haemophilus influenzae, Mycobacterium leprae, Clostridium perfringens, Synechocystis sp. strain PCC6803, and Methanococcus jannaschii. Significant conservation of the amino acid sequence of these proteins in such diverse organisms suggests a very ancient common ancestor for the genes and defines a consensus motif for the phosphoglucosamine mutase active site. We propose renaming the H. pylori ureC gene the glmM gene.     

Identification and characterization of a gene responsible for inhibiting propagation of methylated DNA sequences in mcrA mcrB1 Escherichia coli strains.

Identifying and eliminating endogenous bacterial enzyme systems can significantly increase the efficiency of propagation of eukaryotic DNA in Escherichia coli. We have recently examined one such system which inhibits the propagation of lambda DNA rescued from transgenic mouse tissues. This rescue procedure utilizes lambda packaging extracts for excision of the lambda DNA from the transgenic mouse genome, as well as E. coli cells for subsequent infection and propagation. This assay, in combination with conjugal mating, P1 transduction, and gene cloning, was used to identify and characterize the E. coli locus responsible for this difference in efficiency. It was determined that the E. coli K-12 mcrB gene when expressed on a high-copy-number plasmid can cause a decrease in rescue efficiency despite the presence of the mcrB1 mutation, which inactivates the classic McrB restriction activity. (This mutation was verified by sequence analysis.) However, this McrB1 activity is not observed when the cloned mcrB1 gene is inserted into the E. coli genome at one copy per chromosome. A second locus was identified which causes a decrease in rescue efficiency both when expressed on a high-copy-number plasmid and when inserted into the genome. The data presented here suggest that this locus is mrr and that the mrr gene product can recognize and restrict cytosine-methylated sequences. Removal of this DNA region including the mrr gene from E. coli K-12 strains allows high rescue efficiencies equal to those of E. coli C strains. These modified E. coli K-12 plating strains and lambda packaging extract strains should also allow a significant improvement in the efficiency and representation of eukaryotic genomic and cDNA libraries.     

Gene cloning and characterization of a second alanine racemase from Bacillus subtilis encoded by yncD

Alanine racemase activity was investigated in Bacillus subtilis. A putative second alanine racemase gene (yncD) was cloned in parallel with the previously identified alanine racemase gene, dal. Each of the B. subtilis genes, dal and yncD complemented the Escherichia coli Alr- DadX- double mutant alanine auxotrophic strain MB2159 in vivo, restoring the prototrophic phenotype. Alanine racemase activity was also detected in vitro in cell-free extracts prepared from cultures of E. coli MB2159 harboring plasmids expressing either of the cloned B. subtilis genes and preliminary characterization of enzyme activity is presented.     

The Escherichia coli efg gene and the Rhodobacter capsulatus adgA gene code for NH3-dependent NAD synthetase.

The essential gene efg, which complements ammonia-dependent growth (adgA) mutations in Rhodobacter capsulatus and is located at 38.1 min on the Escherichia coli chromosome, was found to code for NH3-dependent NAD synthetase. Crude extracts from a strain which overproduces the efg gene product contained up to 400 times more activity than crude extracts from the control strain, and the purified Efg protein possessed-NH3-dependent NAD synthetase activity. Glutamine-dependent NAD synthetase activity was found in crude extracts of E. coli but not in the purified enzyme, suggesting that it may be catalyzed by an additional subunit. An R. capsulatus strain carrying an adgA mutation was found to be deficient in NAD synthetase activity, and activity was restored by complementation with the E. coli gene. In accordance with the nomenclature proposed for Salmonella typhimurium (K. T. Hughes, B. M. Olivera, and J. R. Roth, J. Bacteriol. 170:2113-2120, 1988), the efg and adgA genes should now be designated nadE.     

Cloning, nucleotide sequence, overexpression, and inactivation of the Escherichia coli 2-keto-4-hydroxyglutarate aldolase gene.

Having previously determined the complete amino acid sequence of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli (C. J. Vlahos and E. E. Dekker, J. Biol. Chem. 263:11683-11691, 1988), we amplified the gene that codes for this enzyme by the polymerase chain reaction using synthetic degenerate deoxyoligonucleotide primers. The amplified DNA was sequenced by subcloning the polymerase chain reaction products into bacteriophage M13; the nucleotide sequence of the gene was found to be in exact agreement with the amino acid sequence of the gene product. Overexpression of the gene was accomplished by cloning it into the pKK223.3 expression vector so that it was under control of the tac promoter and then using the resultant plasmid, pDP6, to transform E. coli DH5 alpha F'IQ. When this strain was grown in the presence of isopropyl beta-D-thiogalactopyranoside, aldolase specific activity in crude extracts was 80-fold higher than that in wild-type cells and the enzyme constituted approximately 30% of the total cellular protein. All properties of the purified, cloned gene product, including cross-reactivity with antibodies elicited against the wild-type enzyme, were identical with the aldolase previously isolated and characterized. A strain of E. coli in which this gene is inactivated was prepared for the first time by insertion of the kanamycin resistance gene cartridge into the aldolase chromosomal gene.     

Characterization of the Bacillus subtilis ywtD gene,whose product is involved in gamma-polyglutamic acid degradation

The ywtD gene, which codes for an enzyme that degrades gamma-polyglutamic acid (PGA), was cloned from Bacillus subtilis IFO16449. The gene is located immediately downstream of ywsC and ywtABC, a PGA operon involved in PGA biosynthesis, and it showed partial similarity to genes coding for DL-endopeptidase, a peptidoglycan-degrading enzyme. The ywtD gene, from which signal sequence is excised, was inserted into pET15b, and the recombinant plasmid was then transformed into Escherichia coli. Histidine-tagged YwtD was purified from sonicated cells of the transformant. The purified YwtD degraded PGA to yield two hydrolyzed products, a high-molecular-mass product (490 kDa with nearly 100% L-glutamic acid) and an 11-kDa product (with D-glutamic acid and L-glutamic acid in an 80:20 ratio). This finding and results of enzymatic analysis of the two products with carboxypeptidase G suggest that YwtD is a novel enzyme cleaving the gamma-glutamyl bond only between D- and L-glutamic acids of PGA, and it may be designated gamma-DL-glutamyl hydrolase.