The Bacillus subtilis ywjI (glpX) Gene Encodes a Class II Fructose-1,6-Bisphosphatase,Functionally Equivalent to the Class III Fbp Enzyme

We present here experimental evidence that the Bacillus subtilis ywjI gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the fbp-encoded class III enzyme, and constitutes with the upstream gene, murAB, an operon transcribed at the same level under glycolytic or gluconeogenic conditions.Under glycolytic growth conditions, unidirectional phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate is catalyzed by the 6-phosphofructokinase (EC 2.7.1.11). Under gluconeogenic growth conditions, the opposite reaction is catalyzed by the fructose-1,6-bisphosphatase (FBPase) (EC 3.1.3.11) and is required for the synthesis of fructose-6-phosphate and derived metabolites, such as cell wall precursors. Escherichia coli possesses two FBPases: the class I FBPase, encoded by fbp, is highly similar to eukaryotic enzymes, and the class II FBPase (GlpX) (3) has homologues in nearly all prokaryotic genera but in only a few eukaryotes (a green alga, an amoeba, and a moss) and a few archaean species (of the Methanosarcina genus). Biochemical, physiological, and genetic studies allowed the characterization of a Bacillus subtilis enzyme which defined a new class of bacterial FBPases (class III) not structurally related to those previously described and found mainly in Firmicutes (5-7). The gene encoding this activity was identified and, although structurally unrelated to the E. coli class I FBPase gene, was also named fbp (8). In E. coli, the major FBPase is the class I Fbp, whereas the class II GlpX seems to play a minor role (3). In other organisms, the major or even the only FBPase belongs to the class II GlpX family: Bacillus cereus possesses two glpX-like genes and no class I or class III FBPase-encoding gene (26); in Mycobacterium tuberculosis, FBPase activity is encoded only by a glpX-like gene, which has been shown to complement an E. coli mutant lacking such activity (18); in Corynebacterium glutamicum, the only FBPase, essential for growth on gluconeogenic carbon sources, belongs to class II (19). It has been shown that a B. subtilis fbp mutant was still able to grow on substrates such as d-fructose, glycerol, or l-malate as the sole carbon source, which indicated that this mutant could bypass the FBPase reaction during gluconeogenesis (6). Random mutagenesis (ethyl methanesulfonate treatment) performed with this fbp mutant enabled the definition of a B. subtilis locus (bfd) whose additional mutation prevented growth on gluconeogenic carbon sources, but this locus had not been characterized further (7). Determination of the nucleotide sequence of the whole B. subtilis chromosome (16) led to the identification of a putative gene, ywjI, encoding a protein displaying strong homologies with GlpX family members (e.g., 54% identity and 74% similarity with GlpX from C. glutamicum). This gene has therefore been annotated glpX, encoding a class II FBPase, but such annotation has never been validated by genetic or biochemical experimental evidence. In this work, we present experimental evidence that ywjI indeed encodes a class II FBPase.     

Functional Analysis of Fructose-1,6-Bisphosphatase Isozymes (fbp-I and fbp-II Gene Products) in Cyanobacteria

Synechococcus PCC 7942 contains two fructose-1,6-bisphosphataseisozymes (FBPase-I and FBPase-II), while Synechocystis PCC 6803has only one (FBPase-I) in spite of the occurrence of two FBPaseisozyme genes [Tamoi et al. (1998) Biochim. Biophys. Acta 1383:232]. We now demonstrate that disruption of the gene encodingFBPase-II (fbp-II) with a kanamycin resistance gene cartridgedoes not affect cell growth, Chl content, or CO² assimilationin Synechococcus PCC 7942, and disruption of the gene encodingFBPase-I (fbp-I) is a lethal mutation in both cyanobacteria.Accordingly, it is clear that FBPase-I is necessary to sustainphotosynthesis and gluconeogenesis in cyanobacteria. (Received September 10, 1998; Accepted December 10, 1998)     

Degradation of the Gluconeogenic Enzyme Fructose-1,6-Bisphosphatase is Dependent on the Vacuolar ATPase

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced during glucose starvation. After the addition of glucose, inactivated FBPase is selectively targeted to a novel type of Vid (vacuolar import and degradation) vesicle and then to the vacuole for degradation. To identify proteins involved in this pathway, we screened various libraries for mutants that failed to degrade FBPase. Via these approaches, subunits of the vacuolar H+ ATPase (V-ATPase) have been identified repeatedly. The VATPase has established roles in endocytosis, sorting of carboxypeptidase Y and homotypic vacuole fusion. Here, we show that Stv1p, Vph1p, and other subunits of the VATPase are required for FBPase degradation. VPH1 and V0 domain subunits such as Vma3p were required for both Vid vesicle and vacuole function, as determined by an in vitro fusion assay. However, STV1 was only required for the proper function of the Vid vesicles. We also show that the V1 domain participates in the Vid vesicle to vacuoletrafficking step, since most of the V1 subunits are necessary for Vid vesicle-vacuole fusionto occur. The V0 and V1 domains are assembled following a glucose shift and theassembly is independent of protein kinase A and RAV genes. Assembly of the V0 complexis necessary for FBPase trafficking, since mutants that block the assembly and transport ofV0 out of the ER were defective in FBPase degradation.     

Suppression of Kinetic AMP Cooperativity of Fructose-1,6-Bisphosphatase by Carbamoylation of Lysine 50

Selective treatment of pig kidney fructose 1,6-bisphosphatase with cyanate leads to the formation of an active carbamoylated derivative that shows no cooperative interaction between the AMP-binding sites, but completely retains the sensitivity to the inhibitor. By an exhaustive carbamoylation of the enzyme a derivative is formed that has a complete loss of cooperativity and a decrease of sensitivity to AMP. It was proposed that the observed changes of allosteric properties were due to the chemical modification of two lysine residues per enzyme subunit [Slebe et al. (1983), J. Protein Chem. 2, 437–443]. Studies of the temperature dependence of AMP sensitivity and the interaction with Cibacron Blue Sepharose of carbamoylated fructose 1,6-bisphosphatase derivatives indicate that the lysine residue involved in AMP sensitivity is located at the allosteric AMP site, while the lysine residue involved in AMP cooperativity is at a distinct location. Using [¹⁴C]cyanate, we identified both lysine residues in the primary structure of the enzyme; Lys50 is essential for AMP cooperativity and Lys112 appears to be the reactive residue involved in the AMP sensitivity. According to the fructose 1,6-bisphosphatase crystal structure, Lys50 is strategically positioned at the C1–C2 interface, near the molecular center of the tetramer, and Lys112 is in the AMP-binding site. The results reported here, combined with the structural data of the enzyme, strongly suggest that the C1ndash;C2 interface is critical for the propagation of the allosteric signal among the AMP sites on different subunits.     

Isolation and Characterization of Two Enzymes Capable of Hydrolyzing Fructose-1,6-Bisphosphatase from the Lichen Peltigera rufescens

Two enzymes capable of hydrolyzing fructose-1,6-bisphosphate (FBP) have been isolated from the foliose lichen Peltigera rufescens (Weis) Mudd. These enzymes can be separated using Sephadex G-100 and DEAE Sephacel chromatography. One enzyme has a pH optimum of 6.5, and a substrate affinity of 228 micromolar FBP. This enzyme does not require MgCl₂ for activity, and is inhibited by AMP. The second enzyme has a pH optimum of 9.0, with no activity below pH 7.5. This enzyme responds sigmoidally to Mg²⁺, with half-saturation concentration of 2.0 millimolar MgCl₂, and demonstrates hyperbolic kinetics for FBP (K_m = 39 micromolar). This enzyme is activated by 20 millimolar dithiothreitol, is inhibited by AMP, but is not affected by fructose-2-6-bisphosphate. It is hypothesized that the latter enzyme is involved in the photosynthetic process, while the former enzyme is a nonspecific acid phosphatase.     

枯草芽孢杆菌寡聚—1，6—葡萄糖苷酶基因的克隆及其在大肠杆菌中的表达

本研究用鸟枪法构建了枯草芽孢杆菌（Bacillus subtilis)HB002的基因组文库,经平板法筛选得到了六株能水解合成底物对－硝基苯－α-D－葡萄糖吡喃糖苷的阳性克隆,经鉴定均含克隆了寡聚－1,6－葡萄糖苷酶基因的重组质粒（命名为pHBM001-pHBM006)。选择pHBM003,对其插入片段测序分析,此片段内有一编码561个氨基酸的开放阅读框,该 蛋白质的计算分子量为65.985kD。HB002的寡聚－1,6－葡萄糖苷酶的氨基酸序列与Bacillus sp.和凝结芽孢杆菌（Bacillus coagulans)的寡聚－1,6－葡萄糖苷酶的氨基酸序列一致性分别为81％、67％,相似性分别为89％、79％。从pHBM003中扩增出寡聚－1,6－葡萄糖苷酶基因,克隆到pBV220上,转化大肠杆菌（Escherichia coli)DH5α,得到三个能水解对－硝基苯－α－D－葡萄糖吡喃糖苷的阳性克隆HBM003－1～HBM003－3,将此三个菌株热诱导表达,SDS－PAGE电泳可检测到特异表达的蛋白质,其中HBM003－1、HBM003－2表达的蛋白约66kD,为完整的寡聚－1,6－葡萄糖苷酶,而HBM003－3表达的蛋白质偏小;表达的蛋白质均有寡聚－1,6－葡萄糖苷酶活性。     

Reduced Cytosolic Fructose-1,6-Bisphosphatase Activity Leads to Loss of O(2) Sensitivity in a Flaveria linearis Mutant

The mutant plant of Flaveria linearis characterized by Brown et al. (Plant Physiol. 81: 212-215) was studied to determine the cause of the reduced sensitivity to O₂. Analysis of CO₂ assimilation metabolites of freeze clamped leaves revealed that both 3-phosphoglycerate and ribulose 1,5-bisphosphate were high in the mutant plant relative to F. linearis with normal O₂ sensitivity. The k_cat of ribulose-1,5-bisphosphate carboxylase (RuBPCase) was equal in all plant material tested (range 18-22 s⁻¹) indicating that no tight binding inhibitor was present. The degree of RuBPCase carbamylation was reduced in the mutant plant relative to the wild-type plant. Since 3-phosphoglycerate was high in the mutant plant and photosynthesis did not exhibit properties associated with RuBPCase limitations, we believe that the decarbamylation of RuBPCase was a consequence of another lesion in photosynthesis. Fructose 1,6-bisphosphate and its precursors, such as the triose phosphates, were in high concentration in the mutant plant relative to the wild type. The concentrations of the product of the fructose 1,6-bisphosphatase reaction, fructose 6-phosphate, and its isomer, glucose 6-phosphate, were the same in both plants. We found that the mutant plant had up to 75% less cytosolic fructose 1,6-bisphosphatase activity than the wild type but comparable levels of stromal fructose 1,6-bisphosphatase. We conclude that the reduced fructose-1,6-bisphosphatase activity restricts the mutant plant's capacity for sucrose synthesis and this leads to reduced or reversed O₂ sensitivity.     

Gene Expression After Transformation in Bacillus subtilis

Sensitive assays of histidase activity were used to follow the production of this enzyme as directed by a gene newly introduced into cells of Bacillus subtilis by transformation. Histidase activity can be detected in histidase-negative recipient cells within 1 hr after the addition of deoxyribonucleic acid extracted from histidase-positive donors. Enzyme production continues for one to two additional hours and then ceases. Histidase production in the transformed cells is fully sensitive to catabolite repression. Catabolite repression is rapidly established after transformation of recipient cells that are resistant to this form of regulation.     

Characterization of Fructose 1,6-Bisphosphatase and Sedoheptulose 1,7-Bisphosphatase from the Facultative Ribulose Monophosphate Cycle Methylotroph Bacillus methanolicus

The genome of the facultative ribulose monophosphate (RuMP) cycle methylotroph Bacillus methanolicus encodes two bisphosphatases (GlpX), one on the chromosome (GlpX^C) and one on plasmid pBM19 (GlpX^P), which is required for methylotrophy. Both enzymes were purified from recombinant Escherichia coli and were shown to be active as fructose 1,6-bisphosphatases (FBPases). The FBPase-negative Corynebacterium glutamicum Δfbp mutant could be phenotypically complemented with glpX^C and glpX^P from B. methanolicus. GlpX^P and GlpX^C share similar functional properties, as they were found here to be active as homotetramers in vitro, activated by Mn²⁺ ions and inhibited by Li⁺, but differed in terms of the kinetic parameters. GlpX^C showed a much higher catalytic efficiency and a lower K_m for fructose 1,6-bisphosphate (86.3 s⁻¹ mM⁻¹ and 14 ± 0.5 μM, respectively) than GlpX^P (8.8 s⁻¹ mM⁻¹ and 440 ± 7.6 μM, respectively), indicating that GlpX^C is the major FBPase of B. methanolicus. Both enzymes were tested for activity as sedoheptulose 1,7-bisphosphatase (SBPase), since a SBPase variant of the ribulose monophosphate cycle has been proposed for B. methanolicus. The substrate for the SBPase reaction, sedoheptulose 1,7-bisphosphate, could be synthesized in vitro by using both fructose 1,6-bisphosphate aldolase proteins from B. methanolicus. Evidence for activity as an SBPase could be obtained for GlpX^P but not for GlpX^C. Based on these in vitro data, GlpX^P is a promiscuous SBPase/FBPase and might function in the RuMP cycle of B. methanolicus.     

Identification of a Polymyxin Synthetase Gene Cluster of Paenibacillus polymyxa and Heterologous Expression of the Gene in Bacillus subtilis

Polymyxin, a long-known peptide antibiotic, has recently been reintroduced in clinical practice because it is sometimes the only available antibiotic for the treatment of multidrug-resistant gram-negative pathogenic bacteria. Lack of information on the biosynthetic genes of polymyxin, however, has limited the study of structure-function relationships and the development of improved polymyxins. During whole genome sequencing of Paenibacillus polymyxa E681, a plant growth-promoting rhizobacterium, we identified a gene cluster encoding polymyxin synthetase. Here, we report the complete sequence of the gene cluster and its function in polymyxin biosynthesis. The gene cluster spanning the 40.6-kb region consists of five open reading frames, designated pmxA, pmxB, pmxC, pmxD, and pmxE. The pmxC and pmxD genes are similar to genes that encode transport proteins, while pmxA, pmxB, and pmxE encode polymyxin synthetases. The insertional disruption of pmxE led to a loss of the ability to produce polymyxin. Introduction of the pmx gene cluster into the amyE locus of the Bacillus subtilis chromosome resulted in the production of polymyxin in the presence of extracellularly added l-2,4-diaminobutyric acid. Taken together, our findings demonstrate that the pmx gene cluster is responsible for polymyxin biosynthesis.Since polymyxin was first isolated from Bacillus polymyxa in 1947 (1, 4, 47), at least 15 unique polymyxins have been reported (31, 49). Because of its excellent bactericidal activity against gram-negative bacteria, polymyxin antibiotics (polymyxin B and polymyxin E) were used until early 1970 as therapies against many diseases caused by pathogenic microorganisms. However, because they carried serious side effects, including fever, skin eruption, and pain, and also induced severe nephrotoxicity and neurotoxicity (18, 37), it was rapidly replaced by other, better-tolerated antibiotics. In recent years, its application has been restricted to use as an ointment on local surface wounds.Due to the increased and often unnecessary use of antibiotics, pathogenic microorganisms with resistance to antibiotics have become more widespread (2, 14, 30, 38). Under the limited therapeutic options available to treat multidrug-resistant gram-negative bacteria such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, polymyxins are sometimes the only available active antibiotics and have now become important therapeutic agents (13, 25, 28, 29, 55). Many recent reports have shown that patients infected with multidrug-resistant gram-negative pathogens improved upon treatment with polymyxins (19, 27, 44, 48). In addition, polymyxins have been applied to prevent septic shock by removing circulating endotoxin to polystyrene fibers in an immobilized form (8). Therefore, the clinical value of polymyxin, an antibiotic discovered 6 decades ago, is currently being reappraised. However, until now, we have had a very limited understanding of various characteristics of this agent, especially its biosynthetic genes.To analyze structure-function relationships and to develop improved polymyxins with lowered toxicities, novel polymyxin derivatives must be generated. Recently, total or semisynthesis or modifications of polymyxins was performed chemically or enzymatically, and the resulting products were effectively used for structure-function study (6, 20, 36, 45, 50, 52). There is a limitation to obtaining diverse derivatives by using chemical or enzymatic approaches, however, and this limitation is related to the structural complexity of polymyxin. The basic structure of polymyxin is a cyclic heptapeptide with a tripeptide side chain acylated by a fatty acid at the amino terminus (49). Normally, 6-methyloctanoic acid or 6-methylheptanoic acid is attached to the side chain. This structure favors solubility of polymyxin in both water and organic solvent. Unlike other general ribosomally translated peptides, polymyxin is produced by a nonribosomal peptide synthetase (NRPS) (22, 31). NRPSs are multienzyme complexes that have modular structures (35, 46). A module is a distinct section of the multienzyme that is responsible for the incorporation of one or more specific amino acids into the final product. Each module can be divided into different domains, each of which is responsible for a specific biochemical reaction. Three types of domains, the adenylation (A), thiolation (T; also referred to as the peptidyl carrier protein, PCP), and condensation (C) domains, are essential for nonribosomal peptide synthesis. The A-domain plays a role in the selection and activation of an amino acid monomer, the T-domain is responsible for transportation of substrates and elongation intermediates to the catalytic centers, and the C-domain catalyzes peptide bond formation. In addition to these core domains, there are the thioesterase domain (TE-domain), the epimerization domain (E-domain), and some other modification domains. Many NRPS gene clusters have been reported, but no polymyxin biosynthetic gene cluster has been reported to date.During whole genome sequencing of Paenibacillus polymyxa E681, a plant growth-promoting rhizobacterium, we found a gene cluster encoding polymyxin synthetase. In this study, the complete sequences of the polymyxin synthetase genes and the function of the gene cluster have been identified and analyzed by domain analysis, insertional mutagenesis, and heterologous expression of the genes, as well as by antibacterial assay and liquid chromatography-mass spectrometry (LC/MS) analysis of the strains and their culture supernatants. The genome information and the heterologous expression of the polymyxin synthetase gene cluster will be useful for further studies of the regulation of pmx genes, their structure-function relationships, and the improvement of polymyxins.     

枯草芽孢杆菌碱性蛋白酶基因的克隆和表达

目的:获得碱性蛋白酶基因。方法:用PCR的方法从枯草芽孢杆菌A-109中扩增碱性蛋白酶基因(apr),并进行测序分析,构建表达载体,最后转化大肠杆菌BL21,SDS-聚丙烯酰胺凝胶电泳检测该基因的表达情况。结果:apr基因片段含1092个碱基对。该基因片段核苷酸序列与Bacillus amyloliquefaciens subtilisin DFE precursor有99%的同源性,对应的氨基酸序列与Bacillussp.DJ-4有99%的同源性。apr基因在大肠杆菌BL21中获得表达,并表现出蛋白酶活性。结论:获得了具有活性的新的碱性蛋白酶基因。     

长野芽孢杆菌普鲁兰酶基因在枯草芽孢杆菌中的表达及酶学性质研究

根据NCBI上报道的基因序列设计引物,以长野芽孢杆菌(Bacillus naganoensis)ATCC53909的染色体DNA为模板,PCR扩增普鲁兰酶编码基因pulB。将此基因与表达载体pWB980连接构建重组质粒pWB-pulB,并转化枯草芽孢杆菌WB600。SDS-PAGE结果显示,在100 kD处有特异性条带,经测定重组转化子粗酶液酶活力达10.94 U/mL。酶学性质分析表明,其最适反应温度为60℃,最适反应pH为5.0,且在温度30-60℃及pH4.0-6.0范围内稳定,适合淀粉加工行业的应用。