Glyoxylate conversion by Hyphomicrobium species grown on allantoin as nitrogen source

Glyoxylate, formed as a result of allantoin degradation, is converted by Hyphomicrobium species to glycerate via tartronate semialdehyde. Glyoxylate carboligase and tartronate semialdehyde reductase, the two enzymes involved, are present only in cells grown on allantoin as nitrogen source.     

Metabolism and translocation of allantoin in ureide-producing grain legumes

Transfer of the nitrogen and carbon of allantoin to amino acids and protein of leaflets, stems and petioles, apices, peduncles, pods, and seeds of detached shoots of nodulated cowpea (Vigna unguiculata L. Walp. cv. Caloona) plants was demonstrated following supply of [2-¹⁴C], [1,3-¹⁵N]allantoin in the transpiration stream. Throughout vegetative and reproductive growth all plant organs showed significant ureolytic activity and readily metabolized [2-¹⁴C]allantoin to ¹⁴CO₂. A metabolic pathway for ureide nitrogen utilization via allantoic acid, urea, and ammonia was indicated. Levels of ureolytic activity in extracts from leaves and roots of nodulated cowpea were consistently maintained at higher levels than in non-nodulated, NO₃⁻ grown plants.

[¹⁴C]Ureides were recovered in extracts of aphids (Aphis craccivora and Macrosiphum euphorbieae) feeding at different sites on cowpea plants supplied with [2-¹⁴C]allantoin through the transpiration stream or to the upper surface of single leaflets. The data indicated that the ureides were effectively transferred from xylem or leaf mesophyll to phloem, and then translocated in phloem to fruits, apices, and roots.

     

Comparative Analysis of Denitrifying Activities of Hyphomicrobium nitrativorans,Hyphomicrobium denitrificans,and Hyphomicrobium zavarzinii

Hyphomicrobium spp. are commonly identified as major players in denitrification systems supplied with methanol as a carbon source. However, denitrifying Hyphomicrobium species are poorly characterized, and very few studies have provided information on the genetic and physiological aspects of denitrification in pure cultures of these bacteria. This is a comparative study of three denitrifying Hyphomicrobium species, H. denitrificans ATCC 51888, H. zavarzinii ZV622, and a newly described species, H. nitrativorans NL23, which was isolated from a denitrification system treating seawater. Whole-genome sequence analyses revealed that although they share numerous orthologous genes, these three species differ greatly in their nitrate reductases, with gene clusters encoding a periplasmic nitrate reductase (Nap) in H. nitrativorans, a membrane-bound nitrate reductase (Nar) in H. denitrificans, and one Nap and two Nar enzymes in H. zavarzinii. Concurrently with these differences observed at the genetic level, important differences in the denitrification capacities of these Hyphomicrobium species were determined. H. nitrativorans grew and denitrified at higher nitrate and NaCl concentrations than did the two other species, without significant nitrite accumulation. Significant increases in the relative gene expression levels of the nitrate (napA) and nitrite (nirK) reductase genes were also noted for H. nitrativorans at higher nitrate and NaCl concentrations. Oxygen was also found to be a strong regulator of denitrification gene expression in both H. nitrativorans and H. zavarzinii, although individual genes responded differently in these two species. Taken together, the results presented in this study highlight the potential of H. nitrativorans as an efficient and adaptable bacterium that is able to perform complete denitrification under various conditions.     

Dimethylsulfone as a growth substrate for novel methylotrophic species of Hyphomicrobium and Arthrobacter

Dimethylsulfone is a major product of the chemical oxidation in the atmosphere of the principal biogenic sulfur gas, dimethylsulfide, but no studies have been reported on the mechanisms for its microbiological degradation. Three novel strains of bacteria have been isolated from enrichment cultures provided with dimethylsulfone as the only carbon and energy substrate. These are novel facultatively methylotrophic species of Hyphonmicrobium and Arthobacter, capable of growth on a range of one-carbon substrates. Cell-free extracts contained activities of enzymes necessary for a reductive/oxidative pathway for dimethylsulfone degradation: membrane-bound-dimethylsulfone and dimethylsulfoxide reductases, dimethylsulfide monooxygenase, and methanethiol oxidase. Enzymatic evidence is also presented for the subsequent oxidation of formaldehyde by formaldehyde and formate dehydrogenases in the Hyphomicrobium strain and by a dissimilatory ribulose monophosphate cycle in the Arthrobacter strains. The strains also grew on dimethylsulfoxide and dimethylsulfide, and dimethylsulfide-grown bacteria oxidized dimethylsulfide and dimethylsulfoxide but not dimethylsulfone. Formaldehyde assimilation was effected in the Hyphomicrobium strain by the serine pathway, but enzymes of the ribulose monophosphate cycle for formaldehyde assimilation were present in the Arthrobacter strains grown on dimethylsulfone. In contrast, one of the Arthrobacter strains was shown to switch to the serine pathway during growth on methanol. Growth yields on dimethylsulfone and formaldehyde were consistent with the occurrence of the serine pathway in Hyphomicrobium strain S1 and the ribulose monophosphate cycle in Arthrobacter strain TGA, and with the proposed reductive pathway for dimethylsulfone degradation in both.     

Nuclear Apparatus of Hyphomicrobium

The nuclear apparatus of Hyphomicrobium sp. strain B-522 is examined by various microscopy and radiolabeling techniques to determine its behavior during the reproductive cycle of these bacteria. The young, swarmer cell contains a single nucleoid comprised of a deoxyribonucleic acid (DNA) molecule with a molecular weight of 3.1 x 10(9). After development of the swarmer into a mature mother cell with a hypha and bud, the nucleoid replicates and separates into two daughter nucleoids during the initial stages of bud formation. After further development of the bud, one of the daughter nucleoids in the mother cell is rapidly transferred through the hypha to the bud. Half of the old DNA strands pass to each consecutive generation of daughter cells, but only 43% of the stable ribonucleic acid is transferred. The role which the hypha plays in the developmental cycle of these bacteria is discussed, and a mechanism for nuclear transfer is proposed.     

Chemolithotrophic potential of a Hyphomicrobium species,capable of growth on methylated sulphur compounds

The yield of Hyphomicrobium EG on dimethyl sulphoxide, dimethyl sulphide and methylamine, considering the metabolic pathways of these compounds, suggested that the organism gained energy from the oxidation of the sulphur moiety of the former compounds. Indeed, a comparison of chemostat cultures of Hyphomicrobium EG grown on methylamine in the presence and absence of sulphide or thiosulphate proved this obligate methylotroph to be a chemolithoheterotroph. The apparent Y_sulphide and Y_thiosulphate were comparable, being 8–10 g dry weight/mol. In batch cultures thiosulphate concentrations up to 10 mM had a stimulatory effect on the growth rate of Hyphomicrobium EG, whereas higher concentrations increased the organisms doubling time.Enzyme- and respiration data showed that the organism had constitutive enzymes for the breakdown of dimethyl sulphoxide although they were clearly regulated to need. Addition of sulphide or thiosulphate to methylamine-limited chemostat cultures of Hyphomicrobium EG not only resulted in the induction of enzymes necessary for their breakdown, but also caused the enzymes for dimethyl sulphoxide metabolism, especially methyl mercaptan oxidase, to be induced. The formation of H₂O₂, a product of the latter enzyme, was reflected in the relatively high catalase activities during growth on dimethyl sulphoxide and in the organisms inability to grow on this compound in the presence of a catalase inhibitor.Abbreviations DMSO dimethyl sulphoxide - DMS dimethyl sulphide - MM methyl mercaptan - TMAO trimethylamine N-oxide - D dilution rate - GSH redticed glutathione - DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulphate - PES phenazine ethosulphate - RubPCase ribulose 1,5-bisphosphate carboxylase - PEPCase phosphoenol pyruvate carboxylase - Wurster's blue (TMPD) N,N,N,N-tetramethyl-p-phenylenediamine     

Enzymes of Carbohydrate Metabolism in Thiobacillus species

A study was made of enzymes of carbohydrate metabolism in representative thiobacilli grown with and without glucose. The data show that Thiobacillus perometabolis possesses an inducible Entner-Doudoroff pathway and is thus similar to T. intermedius and T. ferrooxidans. T. novellus lacks this pathway. Instead, a non-cyclic pentose phosphate pathway along with the Krebs cycle is apparently the major route of glucose dissimilation in this organism. Glucose does not support or stimulate the growth of strains of T. neapolitanus, T. thioparus, and T. thiooxidans examined, nor does its presence in the growth medium greatly influence their enzymatic constitution. These obligately chemolithotrophic thiobacilli do not possess the Entner-Doudoroff pathway. Their nicotinamide adenine dinucleotide (NAD)-linked isocitrate dehydrogenase activity predominates over their nicotinamide adenine dinucleotide phosphate (NADP)-linked activity; the converse is true for the other thiobacilli. The data suggest that NAD-linked isocitrate dehydrogenase activity in thiobacilli is involved in biosynthetic reactions.     

Biological functions of allantoin

A historical review of allantoin research is presented. An increased allantoin level in the trophoblast and serum of pregnant women has been demonstrated. Allantoin concentration decreased in placental tissues and increased in the serum in developing placental insufficiency.     

Enzymic racemization of allantoin

Allantoin racemase was isolated from cells of Candida utilis, and purified by chromatography on columns of DEAE-cellulose and Sephadex G-100. Using this purified enzyme, the racemization of allantoin in deuterium oxide was investigated. Polarimetric and PMR spectroscopic analyses showed that racemization of allantoin by the enzyme proceeded in parrallel with release of the hydrogen atom (5-H) attached to the asymmetric carbon (C-5) of allantoin. Non-enzymic racemization of allantoin, which was examined for comparison, however, was accompanied by much less or almost no release of allantoin 5-H. This indicates that the mechanism of racemization by the enzyme differs from that of non-enzymic racemization.     

An inducible allantoinase and the specific toxicity of parabanic acid on allantoin utilization in aRhizobium species

ARhizobium sp. (strain NC 92) has been shown to be capable of utilizing uric acid, allantoin, allantoate, urea, and oxaluric acid as sole nitrogen sources. Allantoinase is repressible by NH ₄ ⁺ and inducible by allantoin and, less efficiently, by uric acid, oxaluric acid, and allophanate, but not by urea or parabanic acid. This allantoinase (purified 50-fold to homogeneity) is of 166 Kd M.W., is optimally active at pH 7.5, has a K_m of 4.16 mM and no requirement for sulfhydryl groups or metal ions, and is competitively inhibited by acetohydroxamate (K_i 9 mM). Parabanic acid is nontoxic toRhizobium NC 92 on inorganic N and is highly toxic to growth on allantoin N. Growth inhibition is reversed by supplemented allantoin, and suggestive evidence indicates that NC 92 metabolizes allantoin via the pathway: allantoin allantoate urea NH₃; allophanate is not an intermediate herein. Analysis of allantoinase induction indicates that the mandatory structural requirement is for a free urea moiety in an inducing molecule.     

Polyadenylic acid sequences in the RNA of Hyphomicrobium

Heterogeneous RNA containing polyadenylic acid [poly(A)] sequence has been isolated from Hyphomicrobium by affinity chromatography on oligothymidylic acid cellulose and polyuridylic acid Sepharose columns. About 0.1 to 0.3% of [3H]adenine-labeled RNA over a 60-min period is associated with poly(A) sequences. This percentage decreases to about 0.03 in a 20-h labeling period. The poly(A) tracts recovered after digestion with ribonuclease A and T1 are composed of greater than 95% adenine residues and are up to 200 nucleotides in length with a predominant range of 15 to 40 nucleotides. Adenosine and AMP are present in the ratio of 1:36 in alkaline digests of Hyphomicrobium poly(A) tracts. This is compatible with nucleotide lengths determined on acrylamide gels and location at the 3'-OH terminus of the RNA molecule.     

Cell Wall Composition of Hyphomicrobium Species

Chemical analysis of cell walls obtained from Hyphomicrobium B-522 and from a morphologically and nutritionally distinct organism, Hyphomicrobium neptunium (ATCC 15444), showed that the organisms have a similar cell wall composition, which is typical of gram-negative bacteria. The walls of both strains contained many amino acids, including the characteristic mucopeptide components diaminopimelic acid and muramic acid. Isolation of the mucopeptide by use of sodium dodecyl sulfate was successful only with cell walls of H. neptunium, thus revealing a difference between the walls of the two strains. The mucopeptide preparation contained glucosamine, muramic acid, alanine, glutamic acid, diaminopimelic acid, and glycine in molar ratios of 1.05:1.21:1.84:1.0:1.04:0.31, respectively. The concentration of glycine was sufficiently high to suggest that it is a mucopeptide component rather than an impurity.     

New bacteriophages active on strains of Hyphomicrobium

Fifty-five lytic bacteriophages isolated from water and soil samples were active on many strains of the genus Hyphomicrobium. The optimal isolation procedure was an adsorption method in which samples from a habitat similar to that of the respective host bacterium were used as the phage inoculum. According to the morphology and nucleic acid type these bacteriophages belonged to different families: Myoviridae (type A1: five phages); Styloviridae (type B1: 33 phages; type B2: eight phages) and Podoviridae (type C1: nine phages). The Styloviridae (type B1) appeared in two morphological variants (tails flexible or rigid). All phages investigated were specific for the genus Hyphomicrobium and were unable to lyse members of other genera of hyphal, budding bacteria (e.g. Hyphomonas, Pedomicrobium, genus D, genus T). The host specificity of 42 phages was tested with 156 Hyphomicrobium strains: 122 strains were lysed by at least one of these phages, but 34 Hyphomicrobium strains were not susceptible. Morphotype B1 phages with identical morphology could be distinguished according to their host-range properties on prophage-containing Hyphomicrobium strains. With regard to differences in morphology and host range, 25 phages were selected for more detailed investigations. From these phages DNA was isolated; the melting transition midpoints (Tm) ranged from 67 to 93 degrees C. The upper and higher values suggested the presence of DNA modifications. Six different adsorption patterns could be distinguished among the Hyphomicrobium phages. Preferred attachment sites were the proximal pole of the mother cell, the hyphal tip, the distal pole of the bud, and the distal pole of the swarmer cell.     

Metabolism of molecular species of sulpholipid in barley leaves

Molecular species of sulpholipid (diacylsulphoquinovosylglycerol) were separated and analysed after incubation of developing barley (Hordeum vulgare) leaves with either (1-¹⁴C]-acetate or [³⁵S]-sulphate. The major endogenous molecular species were the trienoic (42%) and the hexaenoic (39%). However, the combined anenoic, monoenoic and dienoic species, which only accounted for 5% of the mass, represented 80% of the labelled species with either precursor. In one experiment, 90% of this radioactivity was found in the dienoic species. The effect of light on the labelling of the molecular species was examined. Acetate is incorporated primarily into the fatty acids of sulpholipid. Transacylation appears to be important in the interconversion of the molecular species of sulpholipid.     

Metabolism of anthracene by a Rhodococcus species

A Rhodococcus sp. isolated from contaminated river sediment was investigated to determine if the isolate could degrade high molecular mass polycyclic aromatic hydrocarbons. The Rhodococcus sp. was able to utilize anthracene (53%), phenanthrene (31%), pyrene (13%), and fluoranthene (5%) as sole source of carbon and energy, but not naphthalene or chrysene. In a study of the degradation of anthracene by a Rhodococcus sp., the identification of ring-fission products indicated at least two ring-cleavage pathways. One results in the production of 6,7-benzocoumarin, previously shown to be produced chemically from the product of meta cleavage of 1,2-dihydroxyanthracene, a pathway which has been well established in Gram-negative bacteria. The second is an ortho cleavage of 1,2-dihydroxyanthracene that produces 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, a dicarboxylic acid ring-fission product. This represents a novel metabolic pathway only identified in Gram-positive bacteria.