UDP-sugar hydrolase isozymes in Salmonella enterica and Escherichia coli: Silent alleles of ushA in related strains of Group I Salmonella isolates, and of ushB in wild-type and K12 strains of E. coli, indicate recent and early silencing events, respectively

Abstract Escherichia coli contains a single periplasmic UDP-glucose hydrolase (5'-nucleotidase) encoded by ushA. Salmonella enterica , serotype Typhimurium, also contains a single UDP-glucose hydrolase but, in contrast to E. coli , it is membrane-bound and is encoded by the non-homologous ushB gene; Salmonella enterica (Typhimurium) also contains a silent allele of the ushA gene ( ushA⁰ ). In this report, we show that nearly all natural isolates of Salmonella contain both UDP-sugar hydrolases, i.e. they are UshA⁺ UshB⁺. The only exceptions are all from sub-group I ( S. gallinarum, S. pullorum , and most Typhimurium strains), are UshA⁻ UshB⁺, and several have been shown to contain an ushA⁰ allele. These data, together with the fact that these latter strains are closely related genetically, strongly suggests a recent silencing mutation(s). We also report the presence in E. coli K-12, and in natural isolates of E. coli , of a DNA sequence which is homologous to the ushB gene of Salmonella ; since E. coli does not contain UshB activity, we tentatively refer to this sequence as ushB⁰ . Since all E. coli strains investigated are UshB⁻, we conclude that the silencing mutation(s) occured relatively eary following the divergence of Escherichia coli and Salmonella from a common ancestor that was ushA⁺ ushB⁺ .     

Isolation, molecular characterization and expression of the ushB gene of Salmonella typhimurium which encodes a membrane-bound UDP-sugar hydrolase

The UDP-sugar hydrolase of Salmonella typhimurium has previously been reported to be located in both the inner and the outer membrane. We have cloned the gene, designated ushB, encoding this enzyme and determined its nucleotide sequence. No significant sequence homology with the periplasmic UDP-sugar hydrolase of Escherichia coli was found at either the DNA or protein level. However, a sequence is detectable, in the E. coli genome, which weakly hybridizes with a specific ushB probe. Polypeptide analysis has allowed the identification of the Salmonella hydrolase which has an Mr of 28,349 as compared to an Mr of 60,767 for the E. coli hydrolase. Most of the protein (approximately 90%) is located in the inner membrane. Two independent membrane fractionation procedures indicate that the remainder may be associated with the outer membrane. The deduced primary structure indicates the presence of an N-terminal signal peptide, although certain features of the region surrounding the putative processing site indicate that processing may be inefficient, or may not occur. Experiments with several inhibitors of signal peptidase function fail to demonstrate the appearance of a precursor form.     

Molecular cloning and sequencing of the gene for CDP-diglyceride hydrolase of Escherichia coli

Previous work from this laboratory had demonstrated that CDP-diglyceride hydrolase of Escherichia coli is encoded by the cdh gene that maps near minute 88 (Bulawa, C. E., and Raetz, C. R. H. (1984) J. Biol. Chem. 259, 11257-11264). We now report the construction of hybrid plasmids and the sequencing of a 1,243-base pair insert carrying cdh. The further construction of BAL31 deletions of this insert, in conjunction with maxicell experiments and in vitro enzyme assay, has led to the identification of a 756-base pair coding sequence for the cdh polypeptide. The molecular weight of the primary translation product deduced from the DNA sequence of the cdh gene is 28,450, in agreement with maxicell experiments. Parallel purification of the enzyme from extracts of wild-type and overproducing strains confirms the presence of a 27-kDa polypeptide in the overproducer, as judged by polyacrylamide gel electrophoresis of the most purified fractions. Inspection of the DNA sequence reveals a very hydrophobic N-terminal domain that may be either a signal peptide or a special region, anchoring the hydrolase to the membrane. In contrast to the CDP-diglyceride synthetase, the overall amino acid composition of the CDP-diglyceride hydrolase is not extraordinarily hydrophobic. Although both CDP-diglyceride synthetase and CDP-diglyceride hydrolase can transfer the CMP moiety of CDP-diglyceride to a suitable acceptor, the primary structures and mechanisms of action of these two enzymes are very different.     

乙内酰脲水解酶基因在大肠杆菌中的克隆表达

节杆菌BT801的乙内酰脲酶系能够水解5-苄基乙内酰脲生成L-苯丙氨酸,其中乙内酰脲水解酶负责乙内酰脲的水解开环。乙内酰脲水解酶的表达对于乙内酰脲酶的催化机制研究及氨基酸的生物不对称合成都具有重要意义。通过PCR技术扩增得到乙内酰脲水解酶基因(hyuH),置于表达载体pT221的,17启动子下游,将构建的重组质粒引入大肠杆菌BL21(DE3)。SDS-PAGE分析在相对分子量50kD处有一较强的表达带,经薄层扫描分析目的蛋白占全菌蛋白的40％,主要以可溶性形式存在,活性分析表明表达产物具有天然的酶活性。     

以蔗糖为底物利用重组大肠杆菌合成甘露醇

【目的】异型发酵乳酸菌可利用胞内产生的甘露醇脱氢酶将果糖高效转化为甘露醇,但果糖作为底物相对昂贵,不利于工业化生产。为了降低生产成本,必须选择廉价的底物。蔗糖相对便宜,并且大量存在于自然界中,能够被重组大肠杆菌利用产生甘露醇。蔗糖水解酶(Sucrose hydrolase)和甘露醇脱氢酶(Mannitol dehydrogenase)是发酵生产甘露醇中催化蔗糖转化成甘露醇的关键酶,构建蔗糖水解酶和甘露醇脱氢酶共表达菌株并进行相关研究是本文的主旨。【方法】利用PCR方法分别从植物乳杆菌(Lactobacillus plantarum)和布氏乳杆菌(Lactobacillus buchneri)基因组DNA中获得sac A和mdh基因,得到大小分别为1 502 bp和1 032 bp的目的基因,经序列分析后将其连接到表达载体p ET-28a(+)上,得到重组表达载体p ET28a-sac A-mdh。将重组质粒转化到大肠杆菌BL21(DE3)中,并用SDS-PAGE分析目的蛋白的表达情况并测定其酶活。【结果】SDS-PAGE显示表达蛋白的大小亚基分子量分别为55.1 k D和37.8 k D,与预期分子量一致,实现sac A和mdh基因的表达。蔗糖水解酶和甘露醇脱氢酶酶活分别为25.78 U/m L和14.56 U/m L。对重组菌株BL21(DE3)/p ET28a-sac A-mdh进行发酵条件优化,甘露醇质量浓度达到45.19 g/L,总糖转化率为37.66%。【结论】与乳酸菌利用蔗糖发酵生产甘露醇相比,产量提高了6倍,且具有发酵周期短、稳定性高等优点,菌株的成功构建为甘露醇工业化生产奠定了基础。     

Nucleotide sequence and high-level expression of the major Escherichia coli phosphofructokinase

The gene for the major phosphofructokinase enzyme in Escherichia coli, pfkA, has been sequenced. Comparison of the amino acid sequence with other phosphofructokinases showed that this enzyme is related to the Bacillus stearothermophilus and rabbit muscle enzymes, but is different from the second, minor phosphofructokinase found in E. coli. The region which has been sequenced comprises the complete pfkA--tpi interval on the E. coli genetic map. Two other genes have been identified from the nucleotide sequence: a gene for a periplasmic sulphate-binding protein, sbp, and for a membrane-bound enzyme, CDP-diglyceride hydrolase, cdh. This establishes the complete gene arrangement in this region as pfkA-sbp-cdh-tpi. The pfkA gene has been subcloned into a high-copy-number plasmid under the control of a strong, chimaeric promoter which arose as an artefact in the construction of the plasmid gene bank from which the original pfkA recombinant was isolated. A specialised recombinant has been constructed which carries a 1.4 X 10(3)-nucleotide insert containing just the pfkA gene flanked by two HindIII recognition sites providing a simple system for the recloning of this gene into different vectors. This recombinant expresses the enzyme at high levels (40-50% of total cell protein is active, soluble phosphofructokinase). This expression system is now being used to study the enzyme using 'reverse genetics'.     

Catalytic mechanism of C-C hydrolase MhpC from Escherichia coli: kinetic analysis of His263 and Ser110 site-directed mutants

C-C hydrolase MhpC (2-hydroxy-6-keto-nona-1,9-dioic acid 5,6-hydrolase) from Escherichia coli catalyses the hydrolytic C-C cleavage of the meta-ring fission product on the phenylpropionic acid catabolic pathway. The crystal structure of E. coli MhpC has revealed a number of active-site amino acid residues that may participate in catalysis. Site-directed mutants of His263, Ser110, His114, and Ser40 have been analysed using steady-state and stopped-flow kinetics. Mutants H263A, S110A and S110G show 10(4)-fold reduced catalytic efficiency, but still retain catalytic activity for C-C cleavage. Two distinct steps are observed by stopped-flow UV/Vis spectrophotometry, corresponding to ketonisation and C-C cleavage: H263A exhibits very slow ketonisation and C-C cleavage, whereas S110A and S110G exhibit fast ketonisation, an intermediate phase, and slow C-C cleavage. H114A shows only twofold-reduced catalytic efficiency, ruling out a catalytic role, but shows a fivefold-reduced K(M) for the natural substrate, and an ability to process an aryl-containing substrate, implying a role for His114 in positioning of the substrate. S40A shows only twofold-reduced catalytic efficiency, but shows a very fast (500 s(-1)) interconversion of dienol (317 nm) to dienolate (394 nm) forms of the substrate, indicating that the enzyme accepts the dienol form of the substrate. These data imply that His263 is responsible for both ketonisation of the substrate and for deprotonation of water for C-C cleavage, a novel catalytic role in a serine hydrolase. Ser110 has an important but non-essential role in catalysis, which appears not to be to act as a nucleophile. A catalytic mechanism is proposed involving stabilisation of reactive intermediates and activation of a nucleophilic water molecule by Ser110.     

Crystallization and preliminary x-ray analysis of Escherichia coli peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase from Escherichia coli, a monomer of 21 kDa, was overexpressed from its cloned gene pth and crystallized by using polyethylene glycol as precipitant. The crystals are orthorhombic and have unit cell parameters a = 47.24 Å, b = 63.59 Å, and c = 62.57 Å. They belong to space group P2₁2₁2₁ and diffract to better than 1.2 Å resolution. The structure is being solved by multiple isomorphous replacement. © 1997 Wiley-Liss Inc.     

Unexpected elevated production of Aquifex aeolicus cytochrome c555 in Escherichia coli cells lacking disulfide oxidoreductases

Mutant strains of Escherichia coli lacking DsbA, DsbB, or DsbD (proteins required for disulfide bond formation in the periplasm) did not produce mitochondrial or chloroplast cytochromes c, as previously observed for bacterial ones. Unexpectedly, however, cytochrome c(555) (AA c(555)) from a hyperthermophile, Aquifex aeolicus, was produced in the E. coli periplasm without Dsb proteins, three times more than with them. These results indicate that the Dsb proteins are not necessarily required for AA c(555) production in E. coli, possibly because of hyperthermophilic origin compared with the others.     

Accumulation of the lipid A precursor UDP-2,3-diacylglucosamine in an Escherichia coli mutant lacking the lpxH gene

The lpxH gene encodes the UDP-2,3-diacylglucosamine-specific pyrophosphatase that catalyzes the fourth step of lipid A biosynthesis in Escherichia coli. To confirm the function of lpxH, we constructed KB21/pKJB5. This strain contains a kanamycin insertion element in the chromosomal copy of lpxH, complemented by plasmid pKJB5, which is temperature-sensitive for replication and harbors lpxH(+). KB21/pKJB5 grows at 30 degrees C but loses viability at 44 degrees C, demonstrating that lpxH is essential. CDP-diglyceride hydrolase (Cdh) catalyzes the same reaction as LpxH in vitro but is non-essential and cannot compensate for the absence of LpxH. The presence of Cdh in cell extracts interferes with the LpxH assay. We therefore constructed KB25/pKJB5, which contains both an in-frame deletion of cdh and a kanamycin insertion mutation in lpxH, covered by pKJB5. When KB25/pKJB5 cells are grown at 44 degrees C, viability is lost, and all in vitro LpxH activity is eliminated. A lipid migrating with synthetic UDP-2,3-diacylglucosamine accumulates in KB25/pKJB5 following loss of the covering plasmid at 44 degrees C. This material was converted to the expected products, 2,3-diacylglucosamine 1-phosphate and UMP, by LpxH. Pseudomonas aeruginosa contains two proteins with sequence similarity to E. coli LpxH. The more homologous protein catalyzes UDP-2,3-diacylglucosamine hydrolysis in vitro. The corresponding gene complements KB25/pKJB5 at 44 degrees C, but the less homologous gene does not. The accumulation of UDP-2,3-diacylglucosamine in our lpxH mutant is consistent with the observation that the lipid A disaccharide synthase LpxB, the next enzyme in the pathway, cannot condense two UDP-2,3-diacylglucosamine molecules, but instead utilizes UDP-2,3-diacylglucosamine as its donor and 2,3-diacylglucosamine 1-phosphate as its acceptor.     

Chloroform-soluble nucleotides in Escherichia coli. Role of CDP-diglyceride in the enzymatic cytidylylation of phosphomonoester acceptors

CDP-diglyceride, the precursor of all the phospholipids in Escherichia coli, is cleaved in vitro to phosphatidic acid and CMP by a membrane-bound hydrolase. Since the physiological function of CDP-diglyceride hydrolase is unknown, we have explored the possibility that this enzyme acts in vivo as either a phosphatidyl- or cytidylyltransferase. To distinguish between these two alternatives, partially purified hydrolase was incubated with CDP-diglyceride in the presence of 50% H218O. Analysis of the reaction products by 31P NMR showed that 18O is incorporated exclusively into CMP, suggesting that the enzyme is a cytidylyltransferase. This conclusion is further supported by the following experimental results: (i) the hydrolase catalyzes the transfer of CMP from CDP-diglyceride to Pi; (ii) numerous phosphomonoesters, such as glycerol 3-phosphate, phosphoserine, and glucose 1-phosphate also function as CMP acceptors, but the corresponding compounds lacking the phosphate residues are not substrates for the enzyme; and (iii) CDP-diglyceride hydrolase exchanges [32P]phosphatidic acid for the phosphatidyl moiety of CDP-diglyceride and 32Pi for the beta-phosphate residue of CDP, indicating the involvement of a novel CMP-enzyme complex. These data suggest a biosynthetic role for CDP-diglyceride hydrolase, and extend the possible functions of CDP-diglyceride in the E. coli envelope.     

Initiation of translation at AUC, AUA and AUU codons in Escherichia coli

A truncated form of the HBL murein hydrolase, encoded by the temperate bacteriophage HB-3, was cloned in a pUC-derivative and translated in Escherichia coli using AUC as start codon, as confirmed by biochemical, immunological, and N-terminal analyses. Using site-directed mutagenesis, we have changed this AUC codon into AUA, AUU and AUG codons. The relative translation efficiencies for these triplets were about 5% for AUC and AUU and 7.5% for AUA compared to that of AUG codon. In the same gene arrangement E. coli beta-galactosidase was also translated at moderate efficiency using AUC as initiator.     

The cytochromes of Escherichia coli

Abstract Escherichia coli contains numerous heme-containing proteins when grown either aerobicaly or anaerobically. These cytochrome species are distributed in the cytoplasm, the periplasm, or are bound to the cytoplasmic membrane. They are involved in various physiological functions, including electron transport, oxidative phosphorylation, assimilatory metabolism and detoxification. One dozen unique cytochrome species have been biochemically and/or genetically characterized. They contain one or more of the four heme groups which E. coli is known to produce: protoheme IX, heme c , heme d , and siroheme. The purpose of this articles is to summarize what we know about the structure and function of this collection of heme proteins.     

Factors and markers of virulence in Escherichia coli from human septicemia

A lethal and necrotic factor which causes cell multinucleation in HeLa cell cultures has previously been shown to be coded by the Vir plasmid of Escherichia coli. Using an absorbed rabbit antiserum which neutralized the Vir toxic properties, we have compared the SDS-PAGE immunoblots from laboratory and field strains which either produce or do not produce Vir toxicity. A single band of 110 kDa was found to be specifically associated with vir toxicity in E. coli strains. This antiserum also recognized the 115 kDa protein band which was previously identified as the cytotoxic necretozing factor (CNF) of certain E. coli strains. These results suggest that the toxin coded by the Vir plasmid is a protein of 110 kDa distinct from, but immunologically related to CNF.     

On the monomeric structure and proposed regulatory properties of phosphoenolpyruvate carboxykinase of Escherichia coli.

Phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating)) (EC 4.1.1.49) has been purified to homogeneity from Escherichia coli. The enzyme shows the same molecular weight (ca. 65000) either by sedimentation equilibrium under nondenaturing conditions or by polyacrylamide gel electrophoresis in the presence of detergent, indicating that the enzyme has a monomeric structure. We have confirmed the previous observation that NADH is an inhibitor of this enzyme, but we have failed to detect the previously reported appearance of homotropic cooperativity with respect to substrate binding the presence of this inhibitor. Lack of such homotropic interactions is in harmony with our conclusion that the enzymes is a monomer. Replacement of Mg2+ by Mn2+ in the assay medium lowers the Km for phosphoenolpyruvate by an order of magnitude, but does not affect the characteristics of inhibition by NADH.     

The Escherichia coli gene encoding the UDP-2,3-diacylglucosamine pyrophosphatase of lipid A biosynthesis

UDP-2,3-diacylglucosamine hydrolase is believed to catalyze the fourth step of lipid A biosynthesis in Escherichia coli. This reaction involves pyrophosphate bond hydrolysis of the precursor UDP-2,3-diacylglucosamine to yield 2,3-diacylglucosamine 1-phosphate and UMP. To identify the gene encoding this hydrolase, E. coli lysates generated with individual lambda clones of the ordered Kohara library were assayed for overexpression of the enzyme. The sequence of lambda clone 157[6E7], promoting overproduction of hydrolase activity, was examined for genes encoding hypothetical proteins of unknown function. The amino acid sequence of one such open reading frame, ybbF, is 50.5% identical to a Haemophilus influenzae hypothetical protein and is also conserved in most other Gram-negative organisms, but is absent in Gram-positives. Cell extracts prepared from cells overexpressing ybbF behind the T7lac promoter have approximately 540 times more hydrolase activity than cells with vector alone. YbbF was purified to approximately 60% homogeneity, and its catalytic properties were examined. Enzymatic activity is maximal at pH 8 and is inhibited by 0.01% (or more) Triton X-100. The apparent K(m) for UDP-2,3-diacylglucosamine is 62 microm. YbbF requires a diacylated substrate and does not cleave CDP-diacylglycerol. (31)P NMR studies of the UMP product generated from UDP-2,3-diacylglucosamine in the presence of 40% H(2)180 show that the enzyme attacks the alpha-phosphate group of the UDP moiety. Because ybbF encodes the specific UDP-2,3-diacylglucosamine hydrolase involved in lipid A biosynthesis, it is now designated lpxH.     

Cloning, sequencing, and characterization of Escherichia coli thioesterase II.

The gene (tesB) encoding Escherichia coli thioesterase II, a low-abundance enzyme of unknown physiological function which can hydrolyze a broad range of acyl-CoA thioesters, has been localized by transposon mutagenesis, cloned and sequenced. A two-cistron construct containing both the lac and tesB promoters was used successfully to overexpress the 286-residue polypeptide. The recombinant enzyme constituted up to 25% of the soluble proteins of E. coli and was readily purified to homogeneity as a tetramer of approximately 120,000 Da. Amino-terminal sequence analysis and electrospray ionization mass spectrometry confirmed the identity of the thioesterase and revealed that the amino-terminal formyl-methionine had been removed yielding a subunit species of average molecular mass 31,842 Da. The protein does not contain the GXSXG motif found characteristically in animal thioesterases which function as chain-terminating enzymes in fatty acid synthesis and exhibits no sequence similarity with these or any other known proteins. Activity of the recombinant enzyme was inhibited by iodoacetamide and diethylpyrocarbonate. The carboxamidomethylated residue was identified as histidine 58, and a role for this amino acid in catalysis is suggested. E. coli strains having a large deletion within the genomic tesB gene grew normally but retained a low level of thioesterase activity toward decanoyl-CoA. This residual activity indicates the presence of an additional decanoyl-CoA hydrolase in E. coli. Over-expression of the recombinant enzyme, under control of the lac promoter, did not alter the fatty acids synthesized by E. coli at any stage of cell growth and the physiological role of this enzyme remains an enigma.     

Thymol-triggered lysis of Escherichia coli expressing Lactobacillus phage LL-H muramidase

Effective disruption of Escherichia coli cells is achieved by the intracellularly accumulated recombinant murein hydrolase (Lactobacillus bacteriophage LL-H muramidase) after the addition of 5 mM thymol. Thymol destroys the integrity and electric potential of the cytoplasmic membrane, and as a consequence the muramidase can access and hydrolyze the cell wall murein leading to cell lysis. Lysis occurred within 5 min after the addition of thymol and seemed to be efficient at high culture densities. This lysis method does not require cell harvesting or addition of other cell wall weakening substances or exogenous enzymes. As a cell disruption method, thymol-triggered lysis is as efficient as sonication in the presence of 1% Triton. Furthermore, thymol did not interfere with the purification steps of Mur by expanded bed adsorption chromatography (EBA), suggesting that the lysis method presented here is well suited for large-scale production and purification of intracellular proteins of E. coli. Received 21 April 1998/ Accepted in revised form 5 December 1998     

Co-expression of bacterial hemoglobin overrides high glucose-induced repression of foreign protein expression in Escherichia coli W3110

Co-expression of Vitreoscilla hemoglobin (VHb) can enhance production of foreign proteins in several microorganisms, including Escherichia coli. Production of foreign proteins [green fluorescent protein (GFP) and organophosphorous hydrolase (OPH)] has been examined in two typical industrial E. coli strains, W3110 (a K12 derivative) and BL21 (a B derivative). In particular, we investigated the effects of VHb co-expression and media glucose concentration on target protein production. We employed the nar O(2)-dependent promoter for self-tuning of VHb expression based on the natural changes in dissolved O(2) levels over the duration of culture. Foreign protein production in strain BL21 was decreased by a high glucose concentration but co-expression of VHb had no effect on this. In contrast, co-expression of VHb in strain W3110 overrode the glucose-induced repression and resulted in steady expression of foreign proteins.