Characterization of a restriction-modification system of the thermotolerant methylotroph Bacillus methanolicus.

We report the isolation of a restriction endonuclease, BmeTI, an isoschizomer of BclI, that recognizes the DNA sequence 5' TGATCA 3'. We also report that BmeTI sites are modified to TGm6ATCA. These findings provide the basis for devising strategies to prevent BmeTI restriction of any DNA introduced into Bacillus methanolicus.     

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.     

一株DDT降解菌的筛选、鉴定及降解特性的初步研究

从DDT污染的土壤中筛选具有DDT降解能力的细菌,经过富集培养、分离纯化得到56株细菌,将其接种到基础盐酵母培养基,7d后用紫外分光光度计法初筛得到降解率较高的一株菌,编号为D-1.通过16S rDNA序列分析结合传统分类学方法确定该菌为寡养单胞菌属(Stenotrophomonas sp.)的一株茵.对菌体降解DDT的特性的研究表明,在培养温度为3℃,底物质量浓度为40 mg/L, pH 7.0,摇床转速为200 r/min的条件下,该菌株对DDT降解10d的降解率为69.0%.     

一株DDT降解菌的筛选、鉴定及降解特性的初步研究

从DDT污染的土壤中筛选具有DDT降解能力的细菌, 经过富集培养、分离纯化得到56株细菌, 将其接种到基础盐酵母培养基, 7 d后用紫外分光光度计法初筛得到降解率较高的一株菌, 编号为D-1。通过16S rDNA序列分析结合传统分类学方法确定该菌为寡养单胞菌属(Stenotrophomonas sp.)的一株菌。对菌体降解DDT的特性的研究表明, 在培养温度为30℃, 底物质量浓度为40 mg/L, pH 7.0, 摇床转速为200 r/min的条件下, 该菌株对DDT降解10 d的降解率为69.0%。     

The Enzymes of Carbon Metabolism in the Thermotolerant Bacillar Strain K1

To determine enzymatic activities in the thermotolerant strain K1 (formerly Sulfobacillus thermosulfidooxidans subsp. thermotolerans), it was grown in a mineral medium with (1) thiosulfate and Fe²⁺ or pyrite (autotrophic conditions), (2) Fe²⁺, thiosulfate, and yeast extract or glucose (mixotrophic conditions), and (3) yeast extract (heterotrophic conditions). Cells grown mixo-, hetero-, and autotrophically were found to contain enzymes of the tricarboxylic acid (TCA) cycle, as well as malate synthase, an enzyme of the glyoxylate cycle. Cells grown organotrophically in a medium with yeast extract exhibited the activity of the key enzymes of the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways. The increased content of carbon dioxide (up to 5 vol %) in the auto- and mixotrophic media enhanced the activity of the enzymes involved in the terminal reactions of the TCA cycle and the enzymes of the pentose phosphate pathway. Carbon dioxide is fixed in the Calvin cycle. The highest activity of ribulose bisphosphate carboxylase was detected in cells grown autotrophically at the atmospheric content of CO₂ in the air used for aeration of the growth medium. The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phospho-enolpyruvate carboxytransphosphorylase decreased with increasing content of CO₂ in the medium.     

Characterization of the Enzymes Encoded by the Anthrose Biosynthetic Operon of Bacillus anthracis

Bacillus anthracis spores, the etiological agents of anthrax, possess a loosely fitting outer layer called the exosporium that is composed of a basal layer and an external hairlike nap. The filaments of the nap are formed by trimers of the collagenlike glycoprotein BclA. Multiple pentasaccharide and trisaccharide side chains are O linked to BclA. The nonreducing terminal residue of the pentasaccharide side chain is the unusual sugar anthrose. A plausible biosynthetic pathway for anthrose biosynthesis has been proposed, and an antABCD operon encoding four putative anthrose biosynthetic enzymes has been identified. In this study, we genetically and biochemically characterized the activities of these enzymes. We also used mutant B. anthracis strains to determine the effects on BclA glycosylation of individually inactivating the genes of the anthrose operon. The inactivation of antA resulted in the appearance of BclA pentasaccharides containing anthrose analogs possessing shorter side chains linked to the amino group of the sugar. The inactivation of antB resulted in BclA being replaced with only trisaccharides, suggesting that the enzyme encoded by the gene is a dTDP-β-l-rhamnose α-1,3-l-rhamnosyl transferase that attaches the fourth residue of the pentasaccharide side chain. The inactivation of antC and antD resulted in the disappearance of BclA pentasaccharides and the appearance of a tetrasaccharide lacking anthrose. These phenotypes are entirely consistent with the proposed roles for the antABCD-encoded enzymes in anthrose biosynthesis. Purified AntA was then shown to exhibit β-methylcrotonyl-coenzyme A (CoA) hydratase activity, as we predicted. Similarly, we confirmed that purified AntC had aminotransferase activity and that purified AntD displayed N-acyltransferase activity.Bacillus anthracis, the causative agent of anthrax, is a Gram-positive, rod-shaped soil bacterium that forms spores when deprived of essential nutrients (15). Spore formation begins with an asymmetric septation that divides the developing cell into a forespore compartment and a larger mother cell compartment, each of which contains a copy of the genome. The mother cell then engulfs the forespore and surrounds it with three protective layers: a cortex composed of peptidoglycan, a closely apposed proteinaceous coat, and a loosely fitting exosporium (10). Mother cell lysis releases the mature spore, which is dormant and capable of surviving in harsh environments for many years (17). When spores encounter an aqueous environment containing nutrients, they can germinate and grow as vegetative cells (21).Recently, interest in B. anthracis spores has intensified in response to their use as agents of bioterrorism. Of particular interest has been the outermost layer of the spore, the exosporium, which serves as a semipermeable barrier to potentially harmful macromolecules (8, 25) and as the vital first point of contact with the immune system of an infected host (11, 18, 30). The exosporium of B. anthracis and of closely related species, such as Bacillus cereus and Bacillus thuringiensis, is comprised of a paracrystalline basal layer and an external hairlike nap (1). The basal layer contains approximately 20 different proteins (20, 23), while the filaments of the nap are formed by trimers of a single collagenlike glycoprotein called BclA (2, 26). The central region of BclA contains a large number of GXX repeats, and the region varies in length in naturally occurring strains of B. anthracis, resulting in hairlike naps of differing lengths (22, 27). Most of the GXX repeats are GPT, and many of the threonine residues are glycosylated. Two major oligosaccharide side chains are present, a pentasaccharide and a trisaccharide, and both are linked to the protein through reducing terminal N-acetylgalactosamine (GalNAc) residues (3). Several studies have demonstrated that the oligosaccharides are antigenic and are exposed on the surface of Bacillus anthracis spores (14, 29). This makes them prime targets for both detection devices and immunoprophylaxis.We previously reported our use of hydrazinolysis to release BclA oligosaccharides from exosporium preparations (3). The primary product was a tetrasaccharide that formed as a result of the undesirable loss of the reducing terminal GalNAc residue of the pentasaccharide, a process called “peeling.” We determined that the oligosaccharide consisted of a linear chain of three rhamnose residues with a novel deoxyamino sugar at its nonreducing terminus. This unusual sugar, 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-d-glucose, was given the trivial name anthrose.Rhamnose is the major sugar present in both the trisaccharide and the pentasaccharide, and a four-gene rhamnose biosynthetic operon was previously identified (22). Previously, we proposed a pathway for anthrose biosynthesis (Fig. ​(Fig.1)1) and identified a four-gene operon (Fig. ​(Fig.2)2) that is essential for its biosynthesis (5). An in-frame deletion of the first gene of the operon reduced the amount of anthrose by approximately 50%, whereas the deletion of any one of the other three genes totally abolished anthrose synthesis. Here, we describe the characterization of the altered oligosaccharide side chains of the four deletion mutants. We also cloned several genes that we predicted are involved in anthrose biosynthesis and demonstrated that the gene products possessed the expected biochemical activities.Open in a separate windowFIG. 1.Proposed biosynthetic pathway of anthrose. The pathway utilizes dTDP-4-keto-6-deoxy-α-d-glucose, an intermediate in rhamnose biosynthesis, and methylcrotonyl-CoA, derived from leucine catabolism. (Modified from reference 5.)Open in a separate windowFIG. 2.Anthrose operon and flanking genes. The four genes of the anthrose operon are antA (BAS3322), antB (BAS3321), antC (BAS3320), and antD (BAS3319). The operon is flanked by genes that encode a putative collagenase (BAS3323) and a putative methyltransferase (BAS3318). (Modified from reference 5.)     

A Novel Approach to Isolation and Functional Characterization of Genomic DNA Sequences from the Methylotrophic Yeast Hansenula polymorpha

A novel approach to isolation and functional characterization of the Hansenula polymorpha genes basing on the use of two strains of different origin is described. One of these strains is better suited for the isolation of genomic DNA fragments, while the other is preferable for their functional analysis. Thirty three genomic sequences governing expression of a reporter protein have been isolated. Analysis of the sequence encoding a homolog of the Saccharomyces cerevisiae cofilin revealed two introns. Another isolated DNA fragment encoded a homolog of the S. cerevisiae Vps10p. Disruption of the corresponding gene resulted in secretion of a vacuolar protein, carboxypeptidase Y, into the culture medium.     

Distribution of Tetrahydromethanopterin-Dependent Enzymes in Methylotrophic Bacteria and Phylogeny of Methenyl Tetrahydromethanopterin Cyclohydrolases

The methylotrophic proteobacterium Methylobacterium extorquens AM1 possesses tetrahydromethanopterin (H(4)MPT)-dependent enzymes, which are otherwise specific to methanogenic and sulfate-reducing archaea and which have been suggested to be involved in formaldehyde oxidation to CO(2) in M. extorquens AM1. The distribution of H(4)MPT-dependent enzyme activities in cell extracts of methylotrophic bacteria from 13 different genera are reported. H(4)MPT-dependent activities were detected in all of the methylotrophic and methanotrophic proteobacteria tested that assimilate formaldehyde by the serine or ribulose monophosphate pathway. H(4)MPT-dependent activities were also found in autotrophic Xanthobacter strains. However, no H(4)MPT-dependent enzyme activities could be detected in other autotrophic alpha-proteobacteria or in gram-positive methylotrophic bacteria. Genes encoding methenyl H(4)MPT cyclohydrolase (mch genes) were cloned and sequenced from several proteobacteria. Bacterial and archaeal Mch sequences have roughly 35% amino acid identity and form distinct groups in phylogenetic analysis.     

Enzymes involved in metabolism of extracellular nucleotides and nucleosides: Functional implications and measurement of activities

Abstract

Extracellular nucleotides and nucleosides mediate diverse signaling effects in virtually all organs and tissues. Most models of purinergic signaling depend on functional interactions between distinct processes, including (i) the release of endogenous ATP and other nucleotides, (ii) triggering of signaling events via a series of nucleotide-selective ligand-gated P2X and metabotropic P2Y receptors as well as adenosine receptors and (iii) ectoenzymatic interconversion of purinergic agonists. The duration and magnitude of purinergic signaling is governed by a network of ectoenzymes, including the enzymes of the nucleoside triphosphate diphosphohydrolase (NTPDase) family, the nucleotide pyrophosphatase/phosphodiesterase (NPP) family, ecto-5′-nucleotidase/CD73, tissue-nonspecific alkaline phosphatase (TNAP), prostatic acid phosphatase (PAP) and other alkaline and acid phosphatases, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP). Along with “classical” inactivating ectoenzymes, recent data provide evidence for the co-existence of a counteracting ATP-regenerating pathway comprising the enzymes of the adenylate kinase (AK) and nucleoside diphosphate kinase (NDPK/NME/NM23) families and ATP synthase. This review describes recent advances in this field, with special emphasis on purine-converting ectoenzymes as a complex and integrated network regulating purinergic signaling in such (patho)physiological states as immunomodulation, inflammation, tumorigenesis, arterial calcification and other diseases. The second part of this review provides a comprehensive overview and basic principles of major approaches employed for studying purinergic activities, including spectrophotometric P_i-liberating assays, high-performance liquid chromatographic (HPLC) and thin-layer chromatographic (TLC) analyses of purine substrates and metabolites, capillary electrophoresis, bioluminescent, fluorometric and electrochemical enzyme-coupled assays, histochemical staining, and further emphasizes their advantages, drawbacks and suitability for assaying a particular catalytic reaction.     

Comparative Studies on Induction of Sporulation and Synthesis of Inducible Enzymes in Bacillus subtilis

An attempt was made to determine whether sporulation and inducible enzyme synthesis in Bacillus subtilis are controlled by the same mechanism of catabolite repression. By the use of a thymine-requiring strain, it has been shown that, whereas sporulation remained repressed unless chromosome replication proceeded to completion, the induction of the enzymes histidase, sucrase, and alpha-glucosidase proceeded quite normally in the absence of continued deoxyribonucleic acid synthesis. It is concluded that the mechanism for overcoming the repression of sporulation differs qualitatively from that involved in overcoming the repression of inducible enzyme synthesis. Attempts to isolate pleiotropic mutants that would provide additional support for this contention were unsuccessful. A pleiotropic mutant deficient in phosphoenolpyruvate-dependent phosphotransferase activity sporulated quite well, whereas a mutant presumed deficient in glutamate synthetase sporulated poorly under all conditions.     

Proteogenomic Characterization of Monocyclic Aromatic Hydrocarbon Degradation Pathways in the Aniline-Degrading Bacterium Burkholderia sp. K24

Burkholderia sp. K24, formerly known as Acinetobacter lwoffii K24, is a soil bacterium capable of utilizing aniline as its sole carbon and nitrogen source. Genomic sequence analysis revealed that this bacterium possesses putative gene clusters for biodegradation of various monocyclic aromatic hydrocarbons (MAHs), including benzene, toluene, and xylene (BTX), as well as aniline. We verified the proposed MAH biodegradation pathways by dioxygenase activity assays, RT-PCR, and LC/MS-based quantitative proteomic analyses. This proteogenomic approach revealed four independent degradation pathways, all converging into the citric acid cycle. Aniline and p-hydroxybenzoate degradation pathways converged into the β-ketoadipate pathway. Benzoate and toluene were degraded through the benzoyl-CoA degradation pathway. The xylene isomers, i.e., o-, m-, and p-xylene, were degraded via the extradiol cleavage pathways. Salicylate was degraded through the gentisate degradation pathway. Our results show that Burkholderia sp. K24 possesses versatile biodegradation pathways, which may be employed for efficient bioremediation of aniline and BTX.     

Characterization of the Bacillus subtilis ywsC gene, involved in gamma-polyglutamic acid production.

The genes required for gamma-polyglutamic acid (PGA) production were cloned from Bacillus subtilis IFO16449, a strain isolated from fermented soybeans. There were four open reading frames in the cloned 4.2-kb DNA fragment, and they were almost identical to those in the ywsC and ywtABC genes of B. subtlis 168. Northern blot analysis showed that the four genes constitute an operon. Three genes, ywsC, ywtA, and ywtB, were disrupted to determine which gene plays a central role in PGA biosynthesis. No PGA was produced in Delta ywsC and Delta ywtA strains, indicating that both of these genes are essential for PGA production. To clarify the function of the YwsC protein, histidine-tagged YwsC (YwsC-His) was produced in the Delta ywsC strain and purified from the lysozyme-treated lysate of the transformant by Ni-nitrilotriacetic acid affinity chromatography. Western blot analysis revealed that the YwsC-His protein consists of two subunits, the 44-kDa and 33-kDa proteins, which are encoded by in-phase overlapping in the ywsC gene. (14)C-labeled PGA was synthesized by the purified proteins from L-[(14)C]-glutamate in the presence of ATP and MnCl(2), through an acylphosphate intermediate, indicating that the ywsC gene encodes PGA synthetase (EC 6.3.2), a crucial enzyme in PGA biosynthesis.