The ADP‐glucose pyrophosphorylase from Streptococcus mutans provides evidence for the regulation of polysaccharide biosynthesis in Firmicutes

Streptococcus mutans is the leading cause of dental caries worldwide. The bacterium accumulates a glycogen‐like internal polysaccharide, which mainly contributes to its carionegic capacity. S. mutans has two genes (glgC and glgD) respectively encoding putative ADP‐glucose pyrophosphorylases (ADP‐Glc PPase), a key enzyme for glycogen synthesis in most bacteria. Herein, we report the molecular cloning and recombinant expression of both genes (separately or together) followed by the characterization of the respective enzymes. When expressed individually GlgC had ADP‐Glc PPase activity, whereas GlgD was inactive. Interestingly, the coexpressed GlgC/GlgD protein was one order of magnitude more active than GlgC alone. Kinetic characterization of GlgC and GlgC/GlgD pointed out remarkable differences between them. Fructose‐1,6‐bis‐phosphate activated GlgC by twofold, but had no effect on GlgC/GlgD. Conversely, phospho‐enol‐pyruvate and inorganic salts inhibited GlgC/GlgD without affecting GlgC. However, in the presence of fructose‐1,6‐bis‐phosphate GlgC acquired a GlgC/GlgD‐like behaviour, becoming sensitive to the stated inhibitors. Results indicate that S. mutans ADP‐Glc PPase is an allosteric regulatory enzyme exhibiting sensitivity to modulation by key intermediates of carbohydrates metabolism in the cell. The particular regulatory properties of the S. mutans enzyme agree with phylogenetic analysis, where GlgC and GlgD proteins found in other Firmicutes arrange in distinctive clusters.     

The DNA-binding protein HU from mesophilic and thermophilic bacilli: gene cloning, overproduction and purification.

The major histone-like bacterial protein (HU)-encoding genes (hup) from five different Bacilli have been cloned, sequenced and overexpressed in Escherichia coli. The five Bacilli selected are closely related, but have different optimum growth temperatures: greater than 70 degrees C for Bacillus caldolyticus and B. caldotenax; 60-65 degrees C for B. stearothermophilus (Bst); 37 degrees C for B. subtilis and 30 degrees C for B. globigii. The deduced amino acid (aa) sequences from the three thermophiles are identical. Those from the two mesophiles are also identical and differ from those of the thermophiles at eleven aa positions. The mesophilic proteins have an extra two aa at the C terminus. Cells harbouring plasmids containing the hup genes can produce HU. An efficient purification scheme using cation-exchange chromatography and fast protein liquid chromatography is presented. This gives approx. 30-40 mg of more than 95% pure Bst HU per litre of E. coli culture.     

Arabidopsis thaliana mutants lacking ADP-glucose pyrophosphorylase accumulate starch and wild-type ADP-glucose content: further evidence for the occurrence of important sources, other than ADP-glucose pyrophosphorylase, of ADP-glucose linked to leaf starch biosynthesis

It is widely considered that ADP-glucose pyrophosphorylase (AGP) is the sole source of ADP-glucose linked to bacterial glycogen and plant starch biosynthesis. Genetic evidence that bacterial glycogen biosynthesis occurs solely by the AGP pathway has been obtained with glgC? AGP mutants. However, recent studies have shown that (i) these mutants can accumulate high levels of ADP-glucose and glycogen, and (ii) there are sources other than GlgC, of ADP-glucose linked to glycogen biosynthesis. In Arabidopsis, evidence showing that starch biosynthesis occurs solely by the AGP pathway has been obtained with the starchless adg1-1 and aps1 AGP mutants. However, mounting evidence has been compiled previewing the occurrence of more than one important ADP-glucose source in plants. In attempting to solve this 20-year-old controversy, in this work we carried out a judicious characterization of both adg1-1 and aps1. Both mutants accumulated wild-type (WT) ADP-glucose and approximately 2% of WT starch, as further confirmed by confocal fluorescence microscopic observation of iodine-stained leaves and of leaves expressing granule-bound starch synthase fused with GFP. Introduction of the sex1 mutation affecting starch breakdown into adg1-1 and aps1 increased the starch content to 8-10% of the WT starch. Furthermore, aps1 leaves exposed to microbial volatiles for 10 h accumulated approximately 60% of the WT starch. aps1 plants expressing the bacterial ADP-glucose hydrolase EcASPP in the plastid accumulated normal ADP-glucose and reduced starch when compared with aps1 plants, whereas aps1 plants expressing EcASPP in the cytosol showed reduced ADP-glucose and starch. Moreover, aps1 plants expressing bacterial AGP in the plastid accumulated WT starch and ADP-glucose. The overall data show that (i) there occur important source(s), other than AGP, of ADP-glucose linked to starch biosynthesis, and (ii) AGP is a major determinant of starch accumulation but not of intracellular ADP-glucose content in Arabidopsis.     

Post-translational redox modification of ADP-glucose pyrophosphorylase in response to light is not a major determinant of fine regulation of transitory starch accumulation in Arabidopsis leaves

ADP-glucose pyrophosphorylase (AGP) is a heterotetrameric enzyme comprising two small and two large subunits that catalyze the production of ADP-glucose linked to starch biosynthesis. The current paradigm on leaf starch metabolism assumes that post-translational redox modification of AGP in response to light is a major determinant of fine regulation of transitory starch accumulation. According to this view, under oxidizing conditions occurring during the night the two AGP small subunits (APS1) are covalently linked via an intermolecular disulfide bridge that inactivates the protein, whereas under reducing conditions occurring during the day NADP-thioredoxin reductase C (NTRC)-dependent reductive monomerization of APS1 activates the enzyme. In this work we have analyzed changes in the redox status of APS1 during dark-light transition in leaves of plants cultured under different light intensities. Furthermore, we have carried out time-course analyses of starch content in ntrc mutants, and in aps1 mutants expressing the Escherichia coli redox-insensitive AGP (GlgC) in the chloroplast. We also characterized aps1 plants expressing a redox-insensitive, mutated APS1 (APS1mut) form in which the highly conserved Cys81 residue involved in the formation of the intermolecular disulfide bridge has been replaced by serine. We found that a very moderate, NTRC-dependent APS1 monomerization process in response to light occurred only when plants were cultured under photo-oxidative conditions. We also found that starch accumulation rates during the light in leaves of both ntrc mutants and GlgC-expressing aps1 mutants were similar to those of wild-type leaves. Furthermore, the pattern of starch accumulation during illumination in leaves of APS1mut-expressing aps1 mutants was similar to that of APS1-expressing aps1 mutants at any light intensity. The overall data demonstrate that post-translational redox modification of AGP in response to light is not a major determinant of fine regulation of transitory starch accumulation in Arabidopsis.     

Over-expression of cbaAB genes of Bacillus stearothermophilus produces a two-subunit SoxB-type cytochrome c oxidase with proton pumping activity

We constructed expression plasmids containing cbaAB, the structural genes for the two-subunit cytochrome bo(3)-type cytochrome c oxidase (SoxB type) recently isolated from a Gram-positive thermophile Bacillus stearothermophilus. B. stearothermophilus cells transformed with the plasmids over-expressed an enzymatically active bo(3)-type cytochrome c oxidase protein composed of the two subunits, while the transformed Escherichia coli cells produced an inactive protein composed of subunit I without subunit II. The oxidase over-expressed in B. stearothermophilus was solubilized and purified. The oxidase contained protoheme IX and heme O, as the main low-spin heme and the high-spin heme, respectively. Analysis of the substrate specificity indicated that the high-affinity site is very specific for cytochrome c-551, a cytochrome c that is a membrane-bound lipoprotein of thermophilic Bacillus. The purified enzyme reconstituted into liposomal vesicles with cytochrome c-551 showed H(+) pumping activity, although the efficiency was lower than those of cytochrome aa(3)-type oxidases belonging to the SoxM-type.     

Thermostable dipeptidase from Bacillus stearothermophilus: its purification, characterization, and comparison with aminoacylase

Dipeptidase (dipeptide hydrolase [EC 3.4.13.11]) has been purified to homogeneity and crystallized from the cell extract of Bacillus stearothermophilus IFO 12983. The enzyme has a molecular weight of about 86,000, and is composed of two subunits identical in molecular weight (43,000). The enzyme contains 2 g atoms of zinc per mol of protein. A variety of dipeptides consisting of glycine or only L-amino acids serve as substrates of the enzyme; Km and Vmax values for L-valyl-L-alanine are 0.5 mM and 68.0 units/mg protein, respectively. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturants such as urea and guanidine hydrochloride. The enzyme also catalyzes hydrolysis of several N-acylamino acids with Vmax values 3-30% of those for the hydrolysis of dipeptides. The thermostable dipeptidase shares various properties with bacterial aminoacylase [EC 3.5.1.14]: their subunit molecular weight, metal content and requirement, amino acid composition, and amino acid sequence in the N-terminal region are very similar.     

Nucleotide sequences of genes encoding heat-stable and heat-labile glyceraldehyde-3-phosphate dehydrogenases; amino acid sequence and protein thermostability

The structural gene (gapST) encoding glyceraldehyde-3-phosphate dehydrogenase (GPDH; EC 1.2.1.12) from Bacillus stearothermophilus has been cloned in Escherichia coli using plasmid pBR322 as a vector; the homologous gene (gapCO) from Bacillus coagulans was cloned from a phage lambda library. Expression of the cloned gap genes revealed that, as in the wild-type (wt) organisms, the GPDH from B. stearothermophilus (GPDH-ST) was intrinsically heat stable (hs) and that from B. coagulans (GPDH-CO) heat labile (hl). The cloned gap genes were sequenced and the deduced amino acid (aa) sequences were found to be highly conserved (91.6% homology), despite the large difference in thermostability between these two enzymes. Of the 28 aa which differ between the two proteins, most of which occur in the middle third of the monomeric subunit, 5 aa involve replacement of alanine in the hl GPDH-CO, by proline in the hs GPDH-ST, and are especially interesting in terms of their potential contributions to thermostability. Conservation at the DNA level is equally dramatic, with the two gap genes exhibiting 93.3% nucleotide sequence homology. These highly expressed genes exhibit an equivalent codon bias, which more closely resembles that of highly expressed E. coli genes, than that of B. stearothermophilus genes whether highly or weakly expressed.     

Occurrence of more than one important source of ADPglucose linked to glycogen biosynthesis in Escherichia coli and Salmonella

To explore the possible occurrence of sources, other than GlgC, of ADPglucose linked to bacterial glycogen biosynthesis we characterized Escherichia coli and Salmonella DeltaglgCAP deletion mutants lacking the whole glycogen biosynthetic machinery. These mutants displayed the expected glycogen-less phenotype but accumulated ADPglucose. Importantly, DeltaglgCAP cells expressing the glycogen synthase encoding glgA gene accumulated glycogen. Protein chromatographic separation of crude extracts of DeltaglgCAP mutants and subsequent activity measurement analyses revealed that these cells possess various proteins catalyzing the conversion of glucose-1-phosphate into ADPglucose. Collectively these findings show that enterobacteria possess more than one important source of ADPglucose linked to glycogen biosynthesis.     

Molecular analysis of pDL10 from Acidianus ambivalens reveals a family of related plasmids from extremely thermophilic and acidophilic archaea.

The 7598-bp plasmid pDL10 from the extremely thermophilic, acidophilic, and chemolithoautotrophic Archaeon Acidianus ambivalens was sequenced. It contains 10 open reading frames (ORFs) organized in five putative operons. The deduced amino acid sequence of the largest ORF (909 aa) showed similarity to bacterial Rep proteins known from phages and plasmids with rolling-circle (RC) replication. From the comparison of the amino acid sequences, a novel family of RC Rep proteins was defined. The pDL10 Rep protein shared 45-80% identical residues with homologous protein genes encoded by the Sulfolobus islandicus plasmids pRN1 and pRN2. Two DNA regions capable of forming extended stem-loop structures were also conserved in the three plasmids (48-69% sequence identity). In addition, a putative plasmid regulatory protein gene (plrA) was found, which was conserved among the three plasmids and the conjugative Sulfolobus plasmid pNOB8. A homolog of this gene was also found in the chromosome of S. solfataricus. Single-stranded DNA of both pDL10 strands was detected with a mung bean nuclease protection assay using PCR detection of protected fragments, giving additional evidence for an RC mechanism of replication.     

Identification of regions critically affecting kinetics and allosteric regulation of the Escherichia coli ADP-glucose pyrophosphorylase by modeling and pentapeptide-scanning mutagenesis

ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the enzyme responsible for the regulation of bacterial glycogen synthesis. To perform a structure-function relationship study of the Escherichia coli ADP-Glc PPase enzyme, we studied the effects of pentapeptide insertions at different positions in the enzyme and analyzed the results with a homology model. We randomly inserted 15 bp in a plasmid with the ADP-Glc PPase gene. We obtained 140 modified plasmids with single insertions of which 21 were in the coding region of the enzyme. Fourteen of them generated insertions of five amino acids, whereas the other seven created a stop codon and produced truncations. Correlation of ADP-Glc PPase activity to these modifications validated the enzyme model. Six of the insertions and one truncation produced enzymes with sufficient activity for the E. coli cells to synthesize glycogen and stain in the presence of iodine vapor. These were in regions away from the substrate site, whereas the mutants that did not stain had alterations in critical areas of the protein. The enzyme with a pentapeptide insertion between Leu(102) and Pro(103) was catalytically competent but insensitive to activation. We postulate this region as critical for the allosteric regulation of the enzyme, participating in the communication between the catalytic and regulatory domains.     

Analysis of the Escherichia coli glycogen gene cluster suggests that catabolic enzymes are encoded among the biosynthetic genes

The nucleotide sequences of the Escherichia coli genome between the glycogen biosynthetic genes glgB and glgC, and 1170 bp of DNA which follows glgA have been determined. The region between glgB and glgC contains an open reading frame (ORF) of 1521 bp which we call glgX. This ORF is capable of coding for an M_r 56 684 protein. The deduced amino acid (aa) sequence for the putative product shows significant similarity to the E. coli glycogen branching enzyme, and to several different glucan hydrolases and transferases. The regions of sequence similarity include residues which have been reported to be involved in substrate binding and catalysis by taka-amylase. This suggests that the proposed product may catalyze hydrolysis or glycosyltransferase reactions. The cloned region which follows glgA contains an incomplete ORF (1149 bp), glgY, which appears to encode 383 aa of the N terminus of glycogen phosphorylase, based upon sequence similarity with the enzyme from rabbit muscle (47% identical aa residues) and with maltodextrin phosphorylase from E. coli (37% identical aa residues). Results suggest that neither ORF is required for glycogen biosynthesis. The localization of glycogen biosynthetic and degradative genes together in a cluster may facilitate the regulation of these systems in vivo.     

Comparative Analysis of the glg Operons of Pectobacterium chrysanthemi PY35 and Other Prokaryotes

A chromosomal region of Pectobacterium chrysanthemi PY35 that contains of genes for glycogen synthesis was isolated from a cosmid library. The operon consists of glycogen branching enzyme (glgB), glycogen debranching enzyme (glgX), ADP-glucose pyrophosphorylase (glgC), glycogen synthase (glgA), and glycogen phosphorylase (glgP) genes. Gene organization is similar to that of Escherichia coli. The purified ADP-glucose pyrophosphorylase (GlgC) was activated by fructose 1,6-bisphosphate and inhibited by AMP. The constructed glgX::Omega mutant failed to integrate into the chromosome of P. chrysanthemi by marker exchange. Phylogenetic analysis based on the 16S rDNA and the amino acid sequence of Glg enzymes showed correlation with other bacteria. gamma-Proteobacteria have the glgX gene instead of the bacilli glgD gene in the glg operon. The possible evolutionary implications of the results among the prokaryotes are discussed.     

Purification and Characterization of Thermostable Pullulanase from Bacillus stearothermophilus and Molecular Cloning and Expression of the Gene in Bacillus subtilis

A thermostable pullulanase (alpha-dextrin 6-glucanohydrolase [EC 3.2.1.41]) from a newly isolated Bacillus stearothermophilus strain (TRS128) was purified and characterized. The enzyme hydrolyzed (1-->6)-alpha-d-glucosidic linkages of pullulan to produce maltotriose, and the optimum temperature was 65 degrees C. About 90% of the enzyme activity was retained after treatment at 65 degrees C for 60 min. By using pTB522 as a vector plasmid, the pullulanase gene was cloned and expressed in Bacillus subtilis.     

Nucleotide sequence of the Synechococcus sp. PCC7942 branching enzyme gene (glgB): expression in Bacillus subtilis

The nucleotide sequence of the Synechococcus sp. PCC7942 glgB gene has been determined. The gene contains a single open reading frame (ORF) of 2322 bp encoding a polypeptide of 774 amino acids (aa) with an Mr of 89,206. Extensive sequence similarity exists between the deduced aa sequence of the Synechococcus sp. glgB gene product and that of the Escherichia coli branching enzyme in the middle portions of the proteins (62% identical aa). In contrast, the N-terminal portions shared little homology. The sequenced region which follows glgB contains an ORF encoding 79 aa of the N terminus of a polypeptide that shares extensive sequence similarity (41% identical aa) with human and rat uroporphyrinogen decarboxylase. This suggests that the region downstream from glgB contains the hemE gene and, therefore, that the organization of genes involved in glycogen biosynthesis in Synechococcus sp. is different from that described for E. coli. A fusion gene was constructed between the 5' end of the Bacillus licheniformis penP gene and the Synechococcus sp. glgB gene. The fusion gene was efficiently expressed in the Gram+ micro-organism Bacillus subtilis and specified a branching enzyme with an optimal temperature for activity similar to the wild-type enzyme.     

Replication Mechanism and Sequence Analysis of the Replicon of pAW63, a Conjugative Plasmid from Bacillus thuringiensis

A 5.8-kb fragment of the large conjugative plasmid pAW63 from Bacillus thuringiensis subsp. kurstaki HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMbeta1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMbeta1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMbeta1 and pIP501, and it is suggested that this region contains the bona fide pAW63 ori. The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of the Enterococcus faecalis plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes repC and prgO, which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in Bacillus subtilis and B. thuringiensis and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other B. thuringiensis subsp. kurstaki strains and to a strain of B. thuringiensis subsp. alesti.     

DNA sequence of a Fibrobacter succinogenes mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase) gene.

The DNA sequence of a mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase [EC 3.2.1.73]) gene from Fibrobacter succinogenes cloned in Escherichia coli was determined. The general features of this gene are very similar to the consensus features for other gram-negative bacterial genes. The gene product was processed for export in E. coli. There is a high level of sequence homology between the structure of this glucanase and the structure of a mixed-linkage beta-glucanase from Bacillus subtilis. The nonhomologous region of the amino acid sequence includes a serine-rich region containing five repeats of the sequence Pro-Xxx-Ser-Ser-Ser-Ser-(Ala or Val) which may be functionally related to the serine-rich region observed in Pseudomonas fluorescens cellulase and the serine- and/or threonine-rich regions observed in Cellulomonas fimi endoglucanase and exoglucanase, in Clostridium thermocellum endoglucanases A and B, and in Trichoderma reesei cellobiohydrolase I, cellobiohydrolase II, and endoglucanase I.     

Complete nucleotide sequence and characterization of pSNA1 from pimaricin-producing Streptomyces natalensis that replicates by a rolling circle mechanism

A cryptic plasmid, pSNA1, has been identified in the pimaricin-producing Streptomyces natalensis strain ATCC 27448. pSNA1 has been mapped with restriction endonucleases and its complete nucleotide sequence was determined. The circular DNA molecule is 9367 bp in length and has a 71.3% G+C content. Its estimated copy number is 30. Analysis of the sequence and codon preferences indicated that pSNA1 contains seven open reading frames [encoding peptides larger than 90 amino acid (aa) residues], ORF 1 to ORF 7, located on both strands of pSNA1. ORF 3 codes for a protein (476 aa) that shows high sequence similarity to replication-associated proteins in Streptomyces plasmids known to replicate via the rolling circle mechanism. Accumulation of single-strand intermediates further indicates that pSNA1 replicates via the rolling circle replication model. ORF 1 encodes a polypeptide of 246 aa that shares homology with KorA proteins encoded by other streptomycete plasmids. ORF 4 (SpdA) codes for a protein (161 aa) possibly involved in intramycelial plasmid transfer. Protein encoded by ORF 2 (309 aa) shares homology with a Streptomyces protein (SpdB2) also involved in plasmid spreading.     

Plasmid-mediated expression of the UmuDC mutagenesis proteins in an Escherichia coli strain engineered for human cytochrome P450 1A2-catalyzed activation of aromatic amines.

The mutagenic actions of many chemicals depend on the activities of bacterial "mutagenesis proteins", which allow replicative bypass of DNA lesions. Genes encoding these proteins occur on bacterial chromosomes and plasmids, often in the form of an operon (such as umuDC or mucAB) encoding two proteins. Many bacterial strains used in mutagenicity testing carry mutagenesis protein genes borne on plasmids, such as pKM101. Our objective was to introduce mutagenesis protein function into Escherichia coli strain DJ4309. This strain expresses recombinant human cytochrome P450 1A2 and NADPH-P450 reductase and carries out the metabolic conversion of aromatic and heterocyclic amines into DNA-reactive mutagens. We discovered that many mutagenesis-protein plasmids severely inhibit the response of strain DJ4309 to 2-amino-3,4-dimethylimid-azo[4,5-f]quinoline (MeIQ), a typical heterocyclic amine mutagen. Among many plasmids examined, one, pGY8294, a pSC101 derivative carrying the umuDC operon, did not inhibit MeIQ mutagenesis. Strain DJ4309 pGY8294 expresses active mutagenesis proteins, as shown by its response to mutagens such as 1-nitropyrene and 4-nitroquinoline 1-oxide (4-NQO), and is as sensitive as the parent strain DJ4309 to P450-dependent mutagens, such as MeIQ and 1-aminopyrene.     

Domain swapping between a cyanobacterial and a plant subunit ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the regulatory step in the pathway for synthesis of bacterial glycogen and starch in plants. ADP-Glc PPases from cyanobacteria (homotetramer) and from potato (Solanum tuberosum) tuber (heterotetramer) are activated by 3-phosphoglycerate and inhibited by inorganic orthophosphate. To study the function of two putative domains, chimeric enzymes were constructed. PSSANA contained the N-terminus (292 amino acids) of the potato tuber ADP-Glc PPase small subunit (PSS) and the C-terminus (159 residues) of the Anabaena PCC 7120 enzyme. ANAPSS was the inverse chimera. These constructs were expressed separately or together with the large subunit of the potato tuber ADP-Glc PPase (PLS), to obtain homo- and heterotetrameric chimeric proteins. Characterization of these forms showed that the N-terminus determines stability and regulatory redox-dependent properties. The chimeric forms exhibited intermediate 3-phosphoglycerate activation properties with respect to the wild-type homotetrameric enzymes, indicating that the interaction between the putative N- and C-domains determines the affinity for the activator. Characterization of the chimeric heterotetramers showed the functionality of the large subunit, mainly in modulating regulation of the enzyme by the coordinate action of 3-phosphoglycerate and inorganic orthophosphate.