Fermentative Production of Thymidine by a Metabolically Engineered Escherichia coli Strain

Thymidine is an important precursor in the production of various antiviral drugs, including azidothymidine for the treatment of AIDS. Since thymidine-containing nucleotides are synthesized only by the de novo pathway during DNA synthesis, it is not easy to produce a large amount of thymidine biologically. In order to develop a host strain to produce thymidine, thymidine phosphorylase, thymidine kinase, and uridine phosphorylase genes were deleted from an Escherichia coli BL21 strain to develop BLdtu. Since the genes coding for the enzymes related to the nucleotide salvage pathway were disrupted, BLdtu was unable to utilize thymidine or thymine, and thymidine degradation activity was completely abrogated. We additionally expressed T4 thymidylate synthase, T4 nucleotide diphosphate reductase, bacteriophage PBS2 TMP phosphohydrolase, E. coli dCTP deaminase, and E. coli uridine kinase in the BLdtu strain to develop a thymidine-producing strain (BLdtu24). BLdtu24 produced 649.3 mg liter⁻¹ of thymidine in a 7-liter batch fermenter for 24 h, and neither thymine nor uridine was detected. However, the dUTP/dTTP ratio was increased in BLdtu24, which could lead to increased double-strand breakages and eventually to cell deaths during fermentation. To enhance thymidine production and to prevent cell deaths during fermentation, we disrupted a gene (encoding uracil-DNA N-glycosylase) involved in DNA excision repair to suppress the consumption of dTTP and developed BLdtug24. Compared with the thymidine production in BLdtu24, the thymidine production in BLdtug24 was increased by ∼1.2-fold (740.3 mg liter⁻¹). Here, we show that a thymidine-producing strain with a relatively high yield can be developed using a metabolic engineering approach.Thymidine, which is composed of 2-deoxyribose and a thymine base, is a commercially useful precursor in the chemical synthesis of various antiviral drugs, including stavudine and zidovudine (azidothymidine), the active ingredient in a formulation for the treatment of AIDS (18, 19). Because thymidine is required only in DNA synthesis, intracellular thymidine levels are very low and are tightly controlled (40). For the production of precursors for antiviral drugs, thymidine is either biologically produced in a low yield by a few modified microorganisms or chemically synthesized through a very costly process (17, 33, 48, 49). Thus, there is a need for developing a more efficient strain for thymidine production on a large scale.In nature, there are two distinct pathways for dTTP synthesis, the salvage and de novo pathways. The salvage pathway enables the cells to utilize preformed nucleobases and nucleosides for nucleotide synthesis, using thymidine phosphorylase (deoA), uridine phosphorylase (udp), and thymidine kinase (tdk) (Fig. ​(Fig.1)1) (40).Open in a separate windowFIG. 1.Thymidine biosynthetic pathway. The steps engineered in this study are indicated by the bold arrows and lines. Components of the catabolism are as follows: pyrA, carbamoylphosphate synthase; pyrBI, aspartate-carbamoyl transferase; pyrC, dihydroorotase; pyrD, dihydroorotate oxidase; pyrE, orotate phosphoribosyltransferase; pyrF, OMP decarboxylase; pyrG, CTP synthetase; pyrH, UMP kinase; TMPase, TMP phosphohydrolase; nrd, nucleotide diphosphate reductase; tdΔI, T4 thymidylate synthase (intron deleted); thyA, thymidylate synthase; dcd, dCTP deaminase; udk, uridine kinase; deoA, thymidine phosphorylase; tdk, thymidine kinase; udp, uridine phosphorylase; dut, deoxyribonucleotide triphosphatase; ndk, nucleotide diphosphate kinase; tmk, TMP kinase; ung, uracil-DNA N-glycosylase; upp, uracil phosphoribosyl-transferase; cdd, cytidine deaminase; codA, cytosine deaminase.As the name indicates, the de novo pathway enables the cells to synthesize nucleobases de novo. The de novo pathway leading to thymidine biosynthesis starts with the condensation of aspartate and carbamoylphosphate, synthesized by carbamoylphosphate synthase (pyrA) (41). This condensation reaction is catalyzed by aspartate-carbamoyl transferase (pyrBI) to produce carbamoyl aspartate, which undergoes several reactions to produce UMP, the common precursor for the synthesis of the pyrimidine ribonucleoside and deoxynucleosides (Fig. ​(Fig.1)1) (39-41). For thymidine biosynthesis, UMP is converted to UDP in a reaction catalyzed by UMP kinase (pyrH), and UDP is converted to dUDP by ribonucleoside diphosphate reductase (nrdAB), which is regulated by NTP effectors through binding to specific allosteric sites on ribonucleotide diphosphate reductase (nrdA). Escherichia coli can synthesize dUMP from both dCDP and dUDP. The major pathway involves phosphorylation of dCDP to dCTP, deamination of dCTP to dUTP, and hydrolysis of dUTP to dUMP. Only 20 to 30% of the cellular dUMP is supplied by hydrolysis of dUTP (29, 37). The deamination of dCTP (dcd) is located at a branch point in the pyrimidine metabolic pathway. Because of its importance, dcd is regulated by a positive homotropic cooperativity toward dCTP and by a feedback inhibition by dTTP (29, 31, 40).Deoxyuridine triphosphatase (dUTPase [dut]) is a pyrophosphatase that contains zinc ions (42). dUTPase catalyzes the hydrolysis of dUTP to PP_i and dUMP, a substrate for thymidylate synthase (thyA). Generally, the intracellular concentration of dUTP is <10 nmol per 1 g dry cell weight (DCW), and that of dTTP exceeds 500 nmol per 1 g DCW (5, 39, 52). The intracellular dUTP-to-dTTP ratio is increased in dut-deficient mutants, leading to an increased frequency of misincorporation of uracil for thymine in DNA (34). This incorporation is transient only because uracil is removed from DNA via a subsequent excision repair initiated by uracil-DNA N-glycosylase, which is encoded by ung (15, 50). Attempted repair of deoxyuridine residues from DNA without adequate dTTP available to complete the repair reaction can result in multiple single-strand breaks, eventually leading to double-strand breaks (15). Indeed, single- and double-strand breaks accumulate in thymidine-deprived cells (16). In such cells, the loss of uracil glycosylase activity should decrease DNA breaks arising from attempted repair and thereby decrease the toxicity of thymidine depletion.The synthesis of dTMP from dUMP involves the transfer of a methylene group and two reducing equivalents from 5,10-methylenetetrahydrofolate to dUMP, catalyzed by the dimeric enzyme thymidylate synthase (thyA). Even though ThyA catalyzes the committed step for de novo synthesis of dTTP, neither the activity of the enzyme nor the expression of the thyA gene seems to be regulated (2, 3).The general strategy used for the development of a thymidine-overproducing strain involves the alleviation of control mechanisms in key pathways. Several different microorganisms have been modified for thymidine production, including E. coli, Brevibacterium helvolum, and Corynebacterium ammoniagenes, by classical mutagenesis methods, and they were selected based on their capacity to grow on toxic thymidine analogues (30, 33, 48, 49). In these studies, feedback inhibition-resistant variants of thymidine biosynthetic enzymes were obtained by random mutation, and high-producing variants were selected. The most optimum B. helvolum strain obtained by this procedure produced 500 mg liter⁻¹ of thymidine by batch fermentation (33). However, engineered B. helvolum and E. coli mutants also produced thymine, deoxyuridine, and uracil, which are unfavorable for thymidine production since it increases costs during the purification process (30, 33, 48, 49). Furthermore, these thymidine-producing strains have residual thymidine degradation activities, resulting in decreased productivities.Thus, we tried to develop a more efficient thymidine-producing strain by enhancing the de novo pathway leading to thymidine biosynthesis and by disrupting the thymidine salvage pathway. The strategy reported here is based on disrupting genes which encode enzymes involved in thymidine degradation and on expressing foreign genes in the de novo pathway leading to thymidine biosynthesis which encode enzymes that are expected to be less sensitive to feedback inhibition by thymidine than the original enzymes in the host strain. The T4 ribonucleotide diphosphate reductase (nrdAB) operon, T4 thioredoxin (nrdC), T4 thymidylate synthase (td), and PBS2 TMP phosphohydrolase (TMPase) were expressed in an E. coli mutant strain which was modified to block the salvage pathway (deoA, tdk, and udp). In order to increase the influx of dUMP, E. coli dCTP deaminase (dcd), deoxyuridine triphosphatase (dut), and uridine kinase (udk) were expressed with phage-derived genes. We found that the dUTP/dTTP ratio was increased by increasing the level of dUTP in our mutant, leading to the frequent misincorporation of dUTP in DNA. In order to prevent frequent temporary DNA breaks and gaps by excision repair caused by the increased intracellular dUTP/dTTP ratio, uracil-DNA N-glycosylase (ung) was additionally disrupted.     

Current Prospects on Production of Microbial Lipid and Other Value-Added Products Using Crude Glycerol Obtained from Biodiesel Industries

Production of microbial lipids using crude glycerol from the biodiesel industry is reviewed in this paper. Approximately 10 wt.% of crude glycerol is obtained for every batch of biodiesel. The crude glycerol accumulated contains various impurities and hence cannot be used for any commercial applications without further purification. Its conversion via biological and chemical routes into valuable products has been studied by different researchers. Varieties of fungal, yeasts, and algal species have been used to produce microbial lipids from crude glycerol. However, research focus on screening a robust industrial oleaginous strain capable of doing this is still on-going. Due to its chemical similarity to vegetable oils, microbial lipids are considered a potential renewable feedstock for biodiesel production and for applications in food and pharmaceutical industries. Its conversion to polyols and subsequently to biobased polymers is also being explored. The rising price of vegetable oils, increasing energy demands, growing environmental concerns, and availability of crude glycerol as a cheap carbon substrate result in considerable potential for the application of these processes in the future.     

Construction and Application of a luxABCDE Reporter System for Real-Time Monitoring of Enterococcus faecalis Gene Expression and Growth

The present work describes the construction of a novel molecular tool for luciferase-based bioluminescence (BL) tagging of Enterococcus faecalis. To this end, a vector (pSL101) and its derivatives conferring a genetically encoded bioluminescent phenotype on all tested strains of E. faecalis were constructed. pSL101 harbors the luxABCDE operon from pPL2lux and the pREG696 broad-host-range replicon and axe-txe toxin-antitoxin cassette, providing segregational stability for long-term plasmid persistence in the absence of antibiotic selection. The bioluminescent signals obtained from three highly expressed promoters correlated linearly (R(2) > 0.98) with the viable-cell count. We employed lux-tagged E. faecalis strains to monitor growth in real time in milk and urine in vitro. Furthermore, bioluminescence imaging (BLI) was used to visualize the magnitude of the bacterial burden during infection in the Galleria mellonella model system. To our knowledge, pSL101 is the first substrate addition-independent reporter system developed for BLI of E. faecalis and an efficient tool for spatiotemporal tracking of bacterial growth and quantitative determination of promoter activity in real time, noninvasively, in infection model systems.     

Cytotoxic Effect of Hemolytic Culture Supernatant from Enterococcus faecalis on Mouse Polymorphonuclear Neutrophils and Macrophages

We reconfirmed that the LD₅₀S of hemolytic Enterococcus faecalis strains were significantly less than those of nonhemolytic E. faecalis strains in normal mice. Hemolysin produced by E. faecalis lysed human, horse, rabbit, and mouse erythrocytes, but not cow and sheep erythrocytes. Sphingomyelin comprises a part of the lipid composition of the erythrocyte membrane of all mammalian species tested. But phosphatidylcholine exists only in human, horse, rabbit, and mouse. These two lipids inhibited lysis of horse erythrocytes by hemolytic E. faecalis. Phosphatidylcholine is probably the binding component on the membrane of erythrocytes for E. faecalis hemolysin. The hemolytic culture supernatant lysed not only erythrocytes but also mouse polymorphonuclear neutrophils (PMNs) and macrophages.     

Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces

A total of 92 enterococci, isolated from the faeces of minipigs subjected to an in vivo feeding trial, were screened for the production of antimicrobial substances. Bacteriocin production was confirmed for seven strains, of which four were identified as Enterococcus faecalis and three as Enterococcus faecium, on the basis of physiological and biochemical characteristics. The bacteriocins produced by the Ent. faecalis strains showed a narrow spectrum of activity, mainly against other Enterococcus spp., compared with those from the Ent. faecium strains showing a broader spectrum of activity, against indicator strains of Enterococcus spp., Listeria spp., Clostridium spp. and Propionibacterium spp. The bacteriocins of all seven Enterococcus strains were inactivated by alpha-chymotrypsin, proteinase K, trypsin, pronase, pepsin and papain, but not by lipase, lysozyme and catalase. The bacteriocins were heat stable and displayed highest activity at neutral pH. The molecular weight of the bacteriocins, as determined by tricine SDS-PAGE, was approximately 3.4 kDa. Only the strains of Ent. faecalis were found to contain plasmids. PCR detection revealed that the bacteriocins produced by Ent. faecium BFE 1170 and BFE 1228 were similar to enterocin A, whereas those produced by Ent. faecium BFE 1072 displayed homology with enterocin L50A and B.     

News & Notes: Growth Inhibition of Pseudomonas aeruginosa and Enterococcus faecalis by Crude Extractives of Mansonia altisima Timber Sawdust

Crude, aqueous methanol and ethanol extracts of sawdust of Mansonia (Mansonia altisima) timber inhibited Pseudomonas aeruginosa and Enterococcus faecalis. The minimum inhibitory concentration ranged from 4.4 to 9.3 mg/ml, while the minimum bactericidal concentration was 10.0–25.0 mg/ml. Enterococcus faecalis was more susceptible, but no marked difference occurred in the antibacterial effect of methanol and ethanol extracts. The aqueous extract was less active.     

巴斯德毕赤酵母表达系统中甘油阻遏相关基因GR1的分离克隆与鉴定

目的：分离克隆并鉴定巴斯德毕赤酵母表达系统中甘油阻遏相关基因。方法：PCR扩增LacZ基因,克隆至pPLC9载体,构建pPIC9-LacZ表达载体,经Sal I线性化后转化巴斯德毕赤酵母GS115,构建GS115-LacZ模式菌株;用限制酶介导整合（REMI）技术使GS115-LacZ菌体基因组产生随机突变,筛选甘油去阻遏的GS115-LacZ△菌体;Southern blot鉴定GS115-LacZ△基因组,用质粒拯救技术和TAIL-PCR克隆未知基因序列并测序。结果：得到甘油去阻遏的GS115-LacZ△菌体,经Southern blot分析,突变仅发生在1个基因中;通过质粒拯救和TAIL-PCR,分离得到阻遏相关基因GR1,共2863bp,经在线BLAST,发现其编码一种过氧化物酶体自吞噬相关蛋白。结论：分离得到阻遏相关基因GR1,与过氧化物酶体自吞噬相关,提示过氧化物酶体自吞噬相关基因可能对醇氧化酶启动子AOX1的转录活性有影响。     

Enzymatic Biodiesel Production from Sunflower Oil by Candida antarctica Lipase in a Solvent-free System

Methanolysis (transesterification with methanol) of sunflower oil by lipase from Candida antarctica (Novozym 435) in a solvent-free system has been studied. Stepwise as well as continuous methanol feeding was applied to avoid strong substrate inhibition. Glycerol was found to cause strong product inhibition on the enzymatic reaction, therefore glycerol removal by dialysis was investigated using a flat sheet membrane module.     

Enzymatic Biodiesel Production from Sunflower Oil by Candida antarctica Lipase in a Solvent-free System

Methanolysis (transesterification with methanol) of sunflower oil by lipase from Candida antarctica (Novozym 435) in a solvent-free system has been studied. Stepwise as well as continuous methanol feeding was applied to avoid strong substrate inhibition. Glycerol was found to cause strong product inhibition on the enzymatic reaction, therefore glycerol removal by dialysis was investigated using a flat sheet membrane module.     

High-Level Production of Porphyrins in Metabolically Engineered Escherichia coli: Systematic Extension of a Pathway Assembled from Overexpressed Genes Involved in Heme Biosynthesis

Due to their spectroscopic properties porphyrins are of special interest for a variety of applications, ranging from drug development or targeting to material sciences and chemical and biological sensors. Since chemical syntheses are limited in terms of regio- and stereoselective functionalization of porphyrins, a biosynthetic approach with tailored enzyme catalysts offers a promising alternative. In this paper, we describe assembly of the entire heme biosynthetic pathway in a three-plasmid system and overexpression of the corresponding genes with Escherichia coli as a host. Without further optimization, this approach yielded remarkable porphyrin production levels, up to 90 μmol/liter, which is close to industrial vitamin B₁₂ production levels. Different combinations of the genes were used to produce all major porphyrins that occur as intermediates in heme biosynthesis. All these porphyrin intermediates were obtained in high yields. The product spectrum was analyzed and quantified by using high-performance liquid chromatography. Intriguingly, although protoporphyrin IX could be produced at high levels, overexpressed Bacillus subtilis ferrochelatase could not convert this substrate appreciably into heme. However, further investigation clearly revealed a high level of expression of the ferrochelatase and a high level of activity in vitro. These results may indicate that heme has a regulatory impact on the iron uptake of E. coli or that the ferrochelatase is inactive in vivo due to an incompatible enzyme interaction.     

Development of a genomic site for gene integration and expression in Enterococcus faecalis

Enterococcus faecalis, a gram-positive opportunistic pathogen, has become one of the leading causes of nosocomial infections. Normally a resident of the gastrointestinal tract, extensive use of antibiotics has resulted in the rise of E. faecalis strains that are resistant to multiple antibiotics. This, compounded with the ability to easily exchange antibiotic determinants with other bacteria, has made certain E. faecalis infections difficult to treat medically. The genetic toolbox for the study of E. faecalis has expanded greatly in recent years, but has lacked methodology to stably introduce a gene in single copy in a non-disruptive manner for complementation or expression of non-native genes. In this study, we identified a specific site in the genome of E. faecalis OG1RF that can serve as an expression site for a gene of interest. This site is well conserved in most of the sequenced E. faecalis genomes. A vector has also been developed to integrate genes into this site by allelic exchange. Using this system, we complemented an in-frame deletion in eutV, demonstrating that the mutation does not cause polar effects. We also generated an E. faecalis OG1RF strain that stably expresses the green fluorescent protein and is comparable to the parent strain in terms of in vitro growth and pathogenicity in C. elegans and mice. Another major advantage of this new methodology is the ability to express integrated genes without the need for maintaining antibiotic selection, making this an ideal tool for functional studies of genes in infection models and co-culture systems.     

Production of an Engineered Killer Peptide in Nicotiana benthamiana by Using a Potato virus X Expression System

The decapeptide killer peptide (KP) derived from the sequence of a single-chain, anti-idiotypic antibody acting as a functional internal image of a microbicidal, broad-spectrum yeast killer toxin (KT) was shown to exert a strong microbicidal activity against human pathogens. With the aim to exploit this peptide to confer resistance to plant pathogens, we assayed its antimicrobial activity against a broad spectrum of phytopathogenic bacteria and fungi. Synthetic KP exhibited antimicrobial activity in vitro towards Pseudomonas syringae, Erwinia carotovora, Botrytis cinerea, and Fusarium oxysporum. KP was also expressed in plants by using a Potato virus X (PVX)-derived vector as a fusion to the viral coat protein, yielding chimeric virus particles (CVPs) displaying the heterologous peptide. Purified CVPs showed enhanced antimicrobial activity against the above-mentioned plant pathogens and human pathogens such as Staphylococcus aureus and Candida albicans. Moreover, in vivo assays designed to challenge KP-expressing plants (as CVPs) with Pseudomonas syringae pv. tabaci showed enhanced resistance to bacterial attack. The results indicate that the PVX-based display system is a high-yield, rapid, and efficient method to produce and evaluate antimicrobial peptides in plants, representing a milestone for the large-scale production of high-added-value peptides through molecular farming. Moreover, KP is a promising molecule to be stably engineered in plants to confer broad-spectrum resistance to phytopathogens.     

Detection of virulence factors in high-level gentamicin-resistant Enterococcus faecalis and Enterococcus faecium isolates from a Tunisian hospital

Phenotypic and genotypic determination of virulence factors were carried out in 46 high-level gentamicin-resistant (HLGR) clinical Enterococcus faecalis (n=34) and Enterococcus faecium (n=12) isolates recovered from different patients in La Rabta Hospital in Tunis, Tunisia, between 2000 and 2003 (all these isolates harboured the aac(6')-aph(2") gene). The genes encoding virulence factors (agg, gelE, ace, cylLLS, esp, cpd, and fsrB) were analysed by PCR and sequencing. The production of gelatinase and hemolysin, the adherence to caco-2 and hep-2 cells, and the capacity for biofilm formation were investigated in all 46 HLGR enterococci. The percentages of E. faecalis isolates harbouring virulence genes were as follows: gelE, cpd, and ace (100%); fsrB (62%); agg (56%); cylLLS (41.2%); and esp (26.5%). The only virulence gene detected among the 12 HLGR E. faecium isolates was esp (58%). Gelatinase activity was detected in 22 of the 34 E. faecalis isolates (65%, most of them with the gelE+-fsrB+ genotype); the remaining 12 isolates were gelatinase-negative (with the gelE+-fsrB- genotype and the deletion of a 23.9 kb fragment of the fsr locus). Overall, 64% of the cylLLS-containing E. faecalis isolates showed beta-hemolysis. A high proportion of our HLGR E. faecalis isolates, in contrast to E. faecium, showed moderate or strong biofilm formation or adherence to caco-2 and hep-2 cells.     

Enhancing Lipid Production from Crude Glycerol by Newly Isolated Oleaginous Yeasts: Strain Selection, Process Optimization, and Fed-Batch Strategy

High lipid-accumulating yeast Trichosporonoides spathulata was newly isolated using crude glycerol as a sole carbon source. After process optimization in a 5-L bioreactor equipped with pH control and aeration system, T. spathulata produced biomass of 11.3 g/L and lipid of 5.01 g/L with a lipid content of 44.3 % using 10 % (w/v) of crude glycerol supplemented only with 0.5 % (w/v) of ammonium sulfate. A one-stage fed-batch feeding with crude glycerol and ammonium sulfate enhanced biomass and lipid production up to 17.3 and 7.25 g/L, respectively, with a lipid content of 41.9 %, while a two-stage fed-batch feeding with only crude glycerol in the second stage led to a lower biomass of 13.8 g/L but a higher lipid production of 7.78 g/L and a higher lipid content of 56.4 %. The fatty acid composition of produced lipid that is similar to plant oil indicates the high potential use of T. spathulata lipid as biodiesel feedstocks.     

Crystallization and preliminary X-ray crystallographic analysis of Ace: a collagen-binding MSCRAMM from Enterococcus faecalis

Ace is a collagen-binding bacterial cell surface adhesin from Enterococcus faecalis. The collagen-binding domain of Ace (termed Ace40) and its truncated form Ace19 have been crystallized by the vapor-diffusion hanging-drop method. Ace19 was crystallized in two different crystal forms. A complete 1.65 A data set has been collected on the orthorhombic crystal form with unit cell parameters a=38.43 b=48.91 and c=83.73 A. Ace40 was crystallized in the trigonal space group P3(1)21 or P3(2)21 with unit cell parameters a=b=80.24, c=105.91 A; alpha=beta=90 and gamma=120 degrees. A full set of X-ray diffraction data was collected to 2.5 A. Three heavy atom derivative data sets have been successfully obtained for Ace19 crystals and structural analysis is in progress.     

Induction of ermAMR from a Clinical Strain of Enterococcus faecalis by 16-Membered-Ring Macrolide Antibiotics

We cloned the MLS_B resistance determinant by PCR from a clinical isolate of Enterococcus faecalis 373, which is induced more strongly by a 16-membered-ring macrolide, tylosin, than by erythromycin. To elucidate the molecular basis of resistance of E. faecalis 373, we analyzed the cloned gene, designated ermAMR, by site-directed mutagenesis and reporter gene assay. Our results showed that an arginine-to-cysteine change in the seventh codon of the putative leader peptide endowed tylosin with resistance inducibility and that TAAA duplication enabled the control region to express the downstream methylase gene at a drastically increased level.     

Functional characterization and Me ion specificity of a Ca-citrate transporter from Enterococcus faecalis

Secondary transporters of the bacterial CitMHS family transport citrate in complex with a metal ion. Different members of the family are specific for the metal ion in the complex and have been shown to transport Mg(2+)-citrate, Ca(2+)-citrate or Fe(3+)-citrate. The Fe(3+)-citrate transporter of Streptococcus mutans clusters on the phylogenetic tree on a separate branch with a group of transporters found in the phylum Firmicutes which are believed to be involved in anaerobic citrate degradation. We have cloned and characterized the transporter from Enterococcus faecalis EfCitH in this cluster. The gene was functionally expressed in Escherichia coli and studied using right-side-out membrane vesicles. The transporter catalyzes proton-motive-force-driven uptake of the Ca(2+)-citrate complex with an affinity constant of 3.5 microm. Homologous exchange is catalyzed with a higher efficiency than efflux down a concentration gradient. Analysis of the metal ion specificity of EfCitH activity in right-side-out membrane vesicles revealed a specificity that was highly similar to that of the Bacillus subtilis Ca(2+)-citrate transporter in the same family. In spite of the high sequence identity with the S. mutans Fe(3+)-citrate transporter, no transport activity with Fe(3+) (or Fe(2+)) could be detected. The transporter of E. faecalis catalyzes translocation of citrate in complex with Ca(2+), Sr(2+), Mn(2+), Cd(2+) and Pb(2+) and not with Mg(2+), Zn(2+), Ni(2+) and Co(2+). The specificity appears to correlate with the size of the metal ion in the complex.     

Expression,purification and activities of the entire family of intact membrane sensor kinases from Enterococcus faecalis

Two-component signal transduction systems are the main mechanism by which bacteria sense and respond to their environment, and their membrane-located histidine protein kinases generally constitute the sensory components of these systems. Relatively little is known about their fundamental mechanisms and precise nature of the molecular signals sensed, because of the technical challenges of producing sufficient quantities of these hydrophobic membrane proteins. This study evaluated the heterologous production, purification and activities of the 16 intact membrane sensor kinases of Enterococcus faecalis. Following the cloning of the genes into expression plasmid pTTQ18His, all but one kinase was expressed successfully in Escherichia coli inner membranes. Purification of the hexa-histidine ‘tagged’ recombinant proteins was achieved for 13, and all but one were verified as intact. Thirteen intact kinases possessed autophosphorylation activity with no added signal when assayed in membrane vesicles or as purified proteins. Signal testing of two functionally-characterized kinases, FsrC and VicK, was successful examplifying the potential use of in vitro activity assays of intact proteins for systematic signal identification. Intact FsrC exhibited an approximately 10-fold increase in activity in response to a two-fold molar excess of synthetic GBAP pheromone, whilst glutathione, and possibly redox potential, were identified for the first time as direct modulators of VicK activity in vitro. The impact of DTT on VicK phosphorylation resulted in increased levels of phosphorylated VicR, the downstream response regulator, thereby confirming the potential of this in vitro approach for investigations of modulator effects on the entire signal transduction process of two-component systems.     

Molecular cloning, expression, and characterization of a novel endo-alpha-N-acetylgalactosaminidase from Enterococcus faecalis

We report here the molecular cloning, expression and characterization of a novel endo-alpha-N-acetylgalactosaminidase, classified into the GH101 family, from Enterococcus faecalis (endo-EF). The recombinant endo-EF was found to catalyze the liberation of core1-disaccharides (Galbeta1-3GalNAc) from core1-pNP (Galbeta1-3GalNAcalpha-pNP) like other GH101 family enzymes. However, endo-EF seems to differ in specificity from the GH101 enzymes reported to date, because it was able to release trisaccharides from core2-pNP (Galbeta1-3[GlcNAcbeta1-6]GalNAcalpha-pNP) and tetrasaccharides from Gal-core2-pNP (Galbeta1-3[Galbeta1-3GlcNAcbeta1-6]GalNAcalpha-pNP). Interestingly, the enzyme could transfer not only core1-disaccharides but also core2-trisaccharides to alkanols generating alkyl-oligosaccharides. Endo-EF should facilitate O-glycoprotein research.