Cloning of the phosphonoacetate hydrolase gene from Pseudomonas fluorescens 23F encoding a new type of carbon–phosphorus bond cleaving enzyme and its expression in Escherichia coli and Pseudomonas putida

The phnA gene encoding a novel carbon–phosphorus bond cleavage enzyme, phosphonoacetate hydrolase, from Pseudomonas fluorescens 23F was cloned and expressed in Escherichia coli and Pseudomonas putida. It conferred on the latter host the ability to mineralize phosphonoacetate but on the former the ability to utilize it as sole phosphorus source only. The nucleotide and deduced amino acid sequences of the phnA gene showed no significant homology with any data bank accessions.     

Phosphonoacetate biosynthesis: In vitro detection of a novel NADP<Superscript>+</Superscript>-dependent phosphonoacetaldehyde-oxidizing activity in cell-extracts of a marine <Emphasis Type="Italic">Roseobacter</Emphasis>

A novel phosphonoacetaldehyde-oxidizing activity was detected in cell-extracts of the marine bacterium Roseovarius nubinhibens ISM grown on 2-aminoethylphosphonic acid (2-AEP; ciliatine). Extracts also contained 2-AEP transaminase and phosphonoacetate hydrolase activities. These findings indicate the existence of a biological route from 2-AEP via phosphonoacetaldehyde for the production of phosphonoacetate, which has not previously been shown to be a natural product. The three enzymes appear to constitute a previously-unreported pathway for the mineralization of 2-AEP which is a potentially important source of phosphorus in the nutrient-stressed marine environment.     

Inhibition of eucaryotic DNA polymerases by phosphonoacetate and phosphonoformate

Phosphonoacetate was found to be an inhibitor of the DNA polymerase α from three human cells, HeLa, Wi-38, and phytohemagglutinin-stimulated lymphocytes. The inhibition patterns were determined. The apparent inhibition constants (K_ii) were about 30 μm. Thus the DNA polymerase α is 15 to 30 times less sensitive to Phosphonoacetate than the herpesvirus-induced DNA polymerase. The DNA polymerase α from Chinese hamster ovary cells and calf thymus was also inhibited. The DNA polymerases β and γ from the eucaryotic cells were relatively insensitive to phosphonoacetate. The sensitivity of the DNA polymerase α and the relative insensitivity of the DNA polymerase β and γ appeared to be general characteristics of the vertebrate polymerases, DNA polymerases from two other eucaryotic cells, yeast DNA polymerase A and B and tobacco cell DNA polymerase, were inhibited by phosphonoacetate, and to about the same extent as the α-polymerases. Fourteen phosphonate analogs were examined for inhibition of the HeLa DNA polymerase α. Only one, phosphonoformate, was an inhibitor. The mechanism of inhibition for phosphonoformate was analogous to that for phosphonoacetate.     

A comparison of three bacterial phosphonoacetate hydrolases from different environmental sources

Cleavage of the carbon–phosphorus bond of the xenobiotic phosphonoacetate by phosphonoacetate hydrolase represents a novel route for the microbial metabolism of organophosphonates, and is unique in that it is substrate-inducible and its expression is independent of the phosphate status of the cell. The enzyme has previously only been demonstrated in cell extracts of Pseudomonas fluorescens 23F. Phosphonoacetate hydrolase activity is now reported in extracts of environmental Curtobacterium sp. and Pseudomonas sp. isolates capable of the phosphate-insensitive mineralization of phosphonoacetate as the sole source of carbon, energy and phosphorus at concentrations up to 40 mmol l⁻¹ and 100 mmol l⁻¹, respectively. The enzymes in both strains were similarly inducible by phosphonoacetate and had a unique specificity for this substrate. However, they differed significantly from each other, and from the previously described Ps. fluorescens 23F enzyme, in respect of their apparent molecular masses, temperature optima, thermostability, sensitivity to inhibition by chelating agents and by structural analogues of phosphonoacetate, and in their affinities for the substrate.     

A role for carbon catabolite repression in the metabolism of phosphonoacetate by Agromyces fucosus Vs2

A strain of Agromyces fucosus, designated Vs2, metabolized a range of organophosphonate compounds as sole phosphorus sources for growth and metabolized phosphonoacetate as a sole carbon, energy and phosphorus source for growth. With phosphonoacetate as the sole phosphorus source and a pyruvate carbon source, transient phosphate release to the medium was observed, in contrast to cultures grown with glucose and phosphonoacetate, where no phosphate release to the medium was observed. Carbon catabolite repression, specifically by means of inducer exclusion of phosphonoacetate, was proposed as the mechanism responsible, and phosphonoacetate hydrolase enzyme assays carried out on cell extracts confirmed that induced phosphonoacetate hydrolase activities were indeed higher in cells grown on pyruvate with phosphonoacetate as sole phosphorus source. This phenomenon has not previously been demonstrated in vivo, and must represent a significant metabolic control of organophosphonate metabolism. The catabolite repression phenomenon was also evident when A. fucosus grew on 2-aminoethylphosphonate as sole phosphorus source, allowing demonstration of a third mode of control for biodegradation of this compound. Excision of stained zymogram gel pieces, followed by tryptic digestion and mass spectrometric analysis, allowed the identification of phosphonoacetate hydrolase-derived peptides.     

Phosphonate metabolism in Helicobacter pylori

Helicobacter pylori has been shown to degrade two phosphonates, N-phosphonoacetyl-l-aspartate and phosphonoacetate; however, the bacterium does not have any genes homologous to those of the known phosphonate metabolism pathways suggesting that H. pylori may have a novel phosphonate metabolism pathway. Growth of H. pylori on phosphonates was studied and the catabolism of these compounds was measured employing ¹H-nuclear magnetic resonance spectroscopy. The specificity of the catabolic enzymes was elucidated by assaying the degradation of several phosphonates and through substrate competition studies. H. pylori was able to utilise phenylphosphonate as a sole source of phosphate for growth. Three strains of H. pylori showed sigmoidal enzyme kinetics of phenylphosphonate catabolism. Allosteric kinetics were removed when lysates were fractionated into cytosolic and membrane fractions. Catabolic rates increased with the addition of DTT, Mg²⁺ and phosphate and decreased with the addition of EDTA. The physiological properties of H. pylori phosphonate metabolism were characterised and the presence of at least two novel phosphonate catabolism pathways that do not require phosphate starvation growth conditions for activity has been established.     

Phosphonoacetate biosynthesis: in vitro detection of a novel NADP(+)-dependent phosphonoacetaldehyde-oxidizing activity in cell-extracts of the marine Roseovarius nubinhibens ISM

A novel phosphonoacetaldehyde-oxidizing activity was detected in cell-extracts of the marine bacterium Roseovarius nubinhibens ISM grown on 2-aminoethylphosphonic acid (2-AEP; ciliatine). Extracts also contained 2-AEP transaminase and phosphonoacetate hydrolase activities. These findings indicate the existence of a biological route from 2-AEP via phosphonoacetaldehyde for the production of phosphonoacetate, which has not previously been shown to be a natural product. The three enzymes appear to constitute a previously-unreported pathway for the mineralization of 2-AEP which is a potentially important source of phosphorus in the nutrient-stressed marine environment.     

Synthesis of 2′-C-Difluoromethyl Substituted Nucleoside Analogs as Ribonucleoside Replacements in Hammerhead Ribozymes

Abstract

2′-Difluoromethyl modified nucleoside analogs 4 and 9 have been prepared and converted into phosphoramidites for the incorporation into hammerhead ribozymes.     

RT-TGGE as a guide for the successful isolation of phosphonoacetate degrading bacteria

AIMS: Use of molecular techniques for the isolation of bacteria capable of phosphonoacetate mineralization as carbon, phosphorus and energy source. METHODS AND RESULTS: RNA extracts obtained at three different stages of an enrichment selecting for phosphonoacetate degrading bacteria were reverse transcribed using 16S rRNA-specific primers, amplified and analysed by temperature gradient gel electrophoresis (TGGE). This information was used to devise a strategy for the isolation of members of the enrichment that were otherwise difficult to obtain in pure culture. We were able to pull out, in total, four out of the six main microbial cultures that were detected by TGGE. Two of the isolates belonging to Mycobacterium and Agromyces genera were for the first time shown to grow in the presence of phosphonoacetate as sole carbon, phosphorus and energy source releasing almost equimolar levels of inorganic phosphate into the culture medium, and they were shown to exhibit phosphonoacetate hydrolase activity in vitro. CONCLUSIONS: The ubiquity of pseudomonad in degradation processes is more likely a consequence of our ignorance of bacterial requirements and physiology, rather than their possession of unique metabolic properties. SIGNIFICANCE AND IMPACT OF THE STUDY: RT-TGGE analysis can be used to guide the successful isolation of micro-organisms difficult to obtain by culture-dependent methods alone.     

In vitro characterization of a phosphate starvation-independent carbon-phosphorus bond cleavage activity in Pseudomonas fluorescens 23F.

A novel, metal-dependent, carbon-phosphorus bond cleavage activity, provisionally named phosphonoacetate hydrolase, was detected in crude extracts of Pseudomonas fluorescens 23F, an environmental isolate able to utilize phosphonoacetate as the sole carbon and phosphorus source. The activity showed unique specificity toward this substrate; its organic product, acetate, was apparently metabolized by the glyoxylate cycle enzymes of the host cell. Unlike phosphonatase, which was also detected in crude extracts of P. fluorescens 23F, phosphonoacetate hydrolase was inducible only in the presence of its sole substrate and did not require phosphate starvation.     

Carbocyclic Nucleosides with a Modified Cyclopentane Skeleton

Abstract

The aminoalcohol 4 has been converted into carbocyclic nucleoside analogues 2 and 3.     

Cytosolic CP bond cleavage activity in bacterial cells isolated from soil

Three kinds of bacteria (CP1, CP9 and CP10), able to accumulate inorganic phosphate (Pi) in a growth medium containing phosphonoacetate as a sole source of phosphorus, were isolated from two hundred soil samples. CP bond cleavage activity in these strains was determined using extracts prepared from cells grown on a medium containing phosphonoacetate. The activity was not found in cell extracts of CP1. Cell extracts prepared from CP9 catalyzed the liberation of Pi only from phosphonoacetate and 2-aminoethylphosphonate. The cell size of CP10 was abnormally large compared with that of CP1 and CP9, and the extracts of CP10 catalyzed the cleavage of CP bonds in methylphosphonate, phosphonoacetate, phenylphosphonate, 2-amino-ethylphosphonate, 2-amino-4-phosphonobutyrate, glyphosate and in phosphonomycine.     

3′-C-Hydroxymethylthymidine: Synthesis and Incorporation into Oligodeoxynucleotide Analogues

Abstract

The stereoselective synthesis of 3′-C-hydroxymethylthymidine (5) in five steps from thymidine has been accomplished and this nucleoside has been incorporated into oligodeoxynucleotides (ODNs) in different ways.     

α-cycloPNA: 1-Aminocylopentane-1-carboxylic Acid-Derived Peptide Nucleic Acid

Abstract

All four diastereoisomers of 3-thymine-1-(^tbutoxycarbonyl)aminocyclopentane- 1-carboxylic acid have been synthesised from (S)-dimethyl malate and thymine monomer 12 has been incorporated into an α-cycloPNA oligomer.     

Akkermansia与高原牛肺水肿病相关性研究

【目的】本文通过对高原牛胃肠道菌群结构组成的分析,从微生物学角度探讨Akkermansia与高原牛肺水肿病的关系。【方法】本研究以沈阳地区健康娟姗牛为对照,以引进入拉萨半年的健康娟姗牛、拉萨本地健康黄牛以及引进入拉萨半年患肺水肿病的娟姗牛的粪便作为分析样本,采用Illumina MiSeq高通量测序技术测定样本中微生物16S rRNA基因V3–V4区序列,通过比较4种粪便样本菌群组成及丰度的差异,探讨Akkermansia与高原牛肺水肿病的相关性。【结果】Verrucomicrobia中Akkermansia在拉萨本地健康黄牛的胃肠道中的含量显著高于引进入拉萨半年的健康娟姗牛,在引进入拉萨半年患肺水肿病的娟姗牛胃肠道中的含量显著高于引进入拉萨半年的健康娟姗牛。在属水平上,沈阳地区健康娟姗牛胃肠道菌群中Akkermansia丰度占比为0.07%;引进入拉萨半年的健康娟姗牛胃肠道菌群中Akkermansia丰度占比为0.09%;拉萨本地黄牛胃肠道菌群中Akkermansia丰度占比为6.62%,是优势菌属;引进入拉萨半年的患肺水肿病的娟姗牛胃肠道菌群中Akkermansia丰度占比为11.85%,且是第一优势菌属。【结论】首次从微生物学角度探讨Akkermansia与高原牛肺水肿病的关系,为将Akkermansia丰度作为诊断肺水肿病的监测指标提供参考,但具体丰度值还有待进一步研究。     

The Synthesis of 2′-C-Functionalised Nucleosides for Incorporation into Catalytic RNA

Abstract

Five 2′-C-functionalized nucleosides (1–5) have been prepared and incorporated into dinucleoside monophosphates. The effect of the functionality on the stability of the adjacent phosphodiester bond toward hydrolysis by nuclease enzymes and extremes of pH has been assessed.     

Synthesis of an Asymmetrically Substituted AZA Crown Ether as Metal and Amino Acid Binding Site in DNA Conjugates

Abstract

Crown ether 4 as a receptor core for protonated primary amines such as amino acids has been synthesized and incorporated into oligodeoxynucleotides as dangling ends.