Indole-3-acetic acid (IAA) production by Arthrobacter species isolated from Azolla.

Arthrobacter species, isolated from the leaf cavities and the microsporocarps of the aquatic fern species Azolla pinnata and Azolla filiculoides, produced indole-3-acetic acid (IAA) in culture when the precursor tryptophan was added to the medium. No IAA production was detected in the absence of tryptophan. Maximum IAA formation was obtained in the first 2 d of incubation. Part of the tryptophan was transformed to N alpha-acetyl-L-tryptophan.     

Isolation of cyanobacteria from the aquatic fern,Azolla

A procedure has been developed to isolate cyanobacteria from the aquatic fern Azolla. The method is based upon recovery of cyanobacterial bundles from digests of plants and use of this material as a massive inoculum for nitrogen-free media, followed by prolonged incubation in light. The procedure appears to select for those cells capable of growth in vitro. Isolated cyanobacteria were found to resemble Anabaena sp. morphologically but were capable of heterotrophic growth and had high nitrogenase activity when grown on fructose in the dark.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Phosphate uptake, efflux and deficiency in the water fern, Azolla

High phosphorus status (High-P) Azolla mexicana plants (P content 15.5 μmoles g fr wt^?1, doubling time ca. 2.2 d) and Low-P plants with early signs of P-deficiency (P content 6.2 μmoles g fr wt^?1, doubling time ca. 3.2 d) were used to study Pi uptake, efflux and deficiency. When High-P plants were transferred to medium lacking Pi, uptake capacity increased 1.5-fold within 12 h and before any detectable change in growth rate (24–48 h). When High-P and Low-P plants were compared, uptake rates from 0.3–10000 mmoles m^?3 Pi were 2.6–1.7 times higher in Low-P than High-P plants (18–1150 vs 7–665 μmoles g fr wt^?1 h^?1). The relationship of uptake rate to concentration was interpreted as arising from a combined operation of a high- and a low-affinity uptake system. Higher uptake in Low-P plants involved a 3.4-fold increase in V_max (high affinity), no change in K_m (high affinity), and a 1.5 to two-fold increase in both V_max (low affinity) and Km (low affinity). Rates of P efflux into 1–1000 mmoles m^?3 Pi were 1.7 to two times higher from High-P than Low-P plants (12–22 vs 7–11 μmoles g fr wt^?1 h^?1). Below 1 mmole m^?3 Pi, uptake and efflux rates were similar: the equilibrium concentration, at which net uptake was zero, was 0.22 mmoles m^?3 for High-P plants and 0.05 mmoles m^?3 for Low-P plants. Similar results were obtained with A. filiculoides. P transport characteristics of Azolla, a fern, are closely comparable with those of higher plants. Its high P requirement in the field arises from its ecological rather than physiological behaviour. We interpret the field behaviour by exploring the relationship between Azolla growth rate in the field, plant P concentration in the field, Pi transport rates required to support such growth, and Pi concentrations in pond waters. The transport characteristics which must operate in the field match those of Low-P plants in the laboratory, not High-P plants. Thus, Pi uptake in High-P plants should be interpreted as repressed from the normal state, instead of that in Low-P plants being induced.     

A d-specific hydantoin amidohydrolase: properties of the metalloenzyme purified from Arthrobacter crystallopoietes

A d-specific hydantoinase has been purified to homogeneity from Arthrobacter crystallopoietes DSM 20117 with a yield of 5% related to the crude extract. The active enzyme is a tetramer of 257 kDa consisting of four identical subunits, each with a molecular mass of 60 kDa. Incubation of the enzyme with the metal-chelating agent EDTA had no inhibitory effect, while 8-hydroxyquinoline-5-sulfonic acid resulted in a complete and irreversible inactivation. The purified enzyme contains zinc as cofactor, which could be detected by subjection to direct analysis using inductive/coupled plasma-atomic emission spectrometry. The hydantoinase has a wide substrate specificity for the d-selective cleavage of 5-monosubstituted hydantoin derivatives with aliphatic and aromatic side chains. The V_max-value for phenylhydantoin is 217 U/mg, the K_m-value is 8 mM. Dihydrouracil was found to be a natural substrate (V_max=35 U/mg). The N-terminal amino acid sequence of the enzyme shows distinct homologies to other metal-dependent cyclic amidases involved in the nucleotide metabolism especially to dihydropyrimidinases as well as to ureases, l- and unselective hydantoinases. Due to these findings, this enzyme has to be considered as a possible link in the evolution to related l-selective and unselective hydantoinases from the genus of Arthrobacter.     

Cellular fatty acid composition of six fastidious,gramnegative, xylem-limited bacteria from plants

Composition of cellular fatty acids was determined for strains of fastidious, Gramnegative, xylem-limited bacteria causing or associated with Pierce's disease of grapevine, phony disease of peach, plum leaf scorch, stunt of ragweed, elm leaf scorch, and periwinkle wilt. The most abundant fatty acids were straight-chain 150, 160, 170, and 180, unsaturated 161, 181, and unsaturated 17-and 19=carbon homologs. Minor fatty acids included straightchain 120, 140, 190, and 200; an unsaturated 15-carbon homolog; hydroxy-substituted 2-OH 120, 3-OH 120, and 3-OH 140; and branched chain iso-140 and iso-200. Cyclopropane acids were not detected. Physiological age had no effect on fatty acid composition. Class analysis of data indicated relative uniformity within the group. Saturated even-carbon chains comprised 31%–42%, unsaturated acids 41%–52%, saturated odd-carbon chains 10%–18%, hydroxysubstituted chains 2%–7%, and branched-chains 1%–4% of total fatty acids. The ratio of saturated-unsaturated acids ranged from 0.8 to 1.2.     

Removal of lead from solution by the non-viable biomass of the water fern Azolla filiculoides

Non-viable biomass of the aquatic fern, Azolla filiculoides, removed up to 93 mg lead/g biomass from solution. Lead removal varied from 30% of the initial lead concentration at pH 1.5 to approximately 95% at pH values of 3.5 and 4.5. Lead removal decreased to 30% of the initial lead concentration if the lead concentration was initially over 400 mg/l. Lead removal remained at approximately 90% between 10 °C and 50 °C. Biomass concentration (4–8 mg/l) had little effect on lead removal. © Rapid Science Ltd. 1998     

A manganese-dependent dioxygenase from Arthrobacter globiformis CM-2 belongs to the major extradiol dioxygenase family.

Almost all bacterial ring cleavage dioxygenases contain iron as the catalytic metal center. We report here the first available sequence for a manganese-dependent 3,4-dihydroxyphenylacetate (3,4-DHPA) 2,3-dioxygenase and its further characterization. This manganese-dependent extradiol dioxygenase from Arthrobacter globiformis CM-2, unlike iron-dependent extradiol dioxygenases, is not inactivated by hydrogen peroxide. Also, ferrous ions, which activate iron extradiol dioxygenases, inhibit 3,4-DHPA 2,3-dioxygenase. The gene encoding 3,4-DHPA 2,3-dioxygenase, mndD, was identified from an A. globiformis CM-2 cosmid library. mndD was subcloned as a 2.0-kb SmaI fragment in pUC18, from which manganese-dependent extradiol dioxygenase activity was expressed at high levels in Escherichia coli. The mndD open reading frame was identified by comparison with the known N-terminal amino acid sequence of purified manganese-dependent 3,4-DHPA 2,3-dioxygenase. Fourteen of 18 amino acids conserved in members of the iron-dependent extradiol dioxygenase family are also conserved in the manganese-dependent 3,4-DHPA 2,3-dioxygenase (MndD). Thus, MndD belongs to the extradiol family of dioxygenases and may share a common ancestry with the iron-dependent extradiol dioxygenases. We propose the revised consensus primary sequence (G,T,N,R)X(H,A)XXXXXXX(L,I,V,M,F)YXX(D,E,T,N,A)PX(G,P) X(2,3)E for this family. (Numbers in brackets indicate a gap of two or three residues at this point in the sequence.) The suggested common ancestry is also supported by sequence obtained from genes flanking mndD, which share significant sequence identity with xylJ and xylG from Pseudomonas putida.     

Amino acid composition and physiochemical characterization of chondroitinase from Arthrobacter aurescens.

The amino acid and carbohydrate compositions of chondroitinase AC [EC 4.2.2.5] from Arthrobacter aurescens were determined, and its physicochemical properties were examined. 1. The enzyme has been shown to be a glycoprotein containing mannose, glucose, glucosamine, and glucuronic acid (3:5:4:2). 2. Its molecular wieght was estimated to be 76,000 by gel filtration on Sephadex G-200, 75,000-80,000 by SDS disc electrophoresis, and 75,800 by sedimentation veolcity. No subunits were detected in the molecule. 3. The physicochemical properties determined include: sedimentation coefficient (s(o)20, w=5.14 S), diffusion constant (D(o)=6.09 X 10(-7) cm2/sec), frictional ratio (f:f(o)=1.19) and apparent partial specific volume (v=0.73 ml/g). 4. The optical rotatory dispersion and circular dichroism of the enzyme were investigated. The contents of alpha-helix and beta-structure of the enzyme were estimated to be 16 and 25%, respectively.     

Molecular cloning and expression of an isomalto-dextranase gene from Arthrobacter globiformis T6.

The gene encoding an extracellular isomalto-dextranase, designated imd, was isolated from the chromosomal DNA of Arthrobacter globiformis T6 and cloned and expressed in Escherichia coli. A single open reading frame consisting of 1,926 base pairs that encoded a polypeptide composed of a signal peptide of 39 amino acids and a mature protein of 602 amino acids (M(r), 65,900) was found. The primary structure had no significant homology with the structures of any other reported carbohydrases, including two other dextranases. Transformed E. coli cells carrying the 2.3-kb fragment overproduced isomalto-dextranase into the periplasmic space under control of the promoter of the imd gene itself.     

Oxidative pathway of choline to betaine in the soluble fraction prepared from Arthrobacter globiformis.

One strain of bacteria which showed high H2O2-generating activity was isolated from soil and characterized as Arthrobacter globiformis based on its morphological, nutritional, and physiological characteristics. The activities of H2O2 generation, NAD reduction and oxygen consumption in the bacterial cells were examined using choline, betaine aldehyde or betaine as substrate. Choline was oxidized to betaine aldehyde under aerobic conditions in a reaction coupled with H2O2 generation and oxygen consumption. On the other hand, betaine aldehyde seemed to be oxidized to betaine through two distinct oxidative reactions, H2O2 generation (oxygen consumption) under aerobic conditions and NAD reduction under either aerobic or anaerobic conditions. These enzyme activities were found in the supernatant fraction of the sonicated cell preparation.     

Phospholipid composition and fatty acid patterns of isolated phospholipids from rabbit reticulocyte mitochondria

The phospholipid composition and fatty acid patterns of individual phospholipid classes were determined in mitochondria from rabbit reticulocytes. Compared to mitochondria from rat liver reticulocyte, mitochondria exhibit about twice the amount of phospholipids. The phospholipid pattern of reticulocyte mitochondria (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and cardiolipin) is comparable with other mitochondrial species. Mitochondrial fractions from reticulocytes are characterized, however, by an additional content of sphingomyelin. This sphingomyelin differs in its fatty acid composition from the sphingomyelin of the plasma membrane. The fatty acid patterns of all other phospholipids essentially correspond to those of mitochondria from other sources and to those of plasma membranes as well.     

Sphere-Rod Morphogenesis in Arthrobacter crystallopoietes I. Cell Wall Composition and Polysaccharides of the Peptidoglycan

Cell walls of Arthrobacter crystallopoietes were prepared from cells grown as spheres and from peptone- and succinate-induced rod stage cells. Undegraded polysaccharide backbones of the peptidoglycans were isolated from myxobacter AL-1 protease digests by ECTEOLA cellulose and Sephadex G-50 chromatography. The polysaccharide backbones of the sphere cell wall peptidoglycan are heterogeneous in their size, and average less than 40 hexosamines per chain. Those of the rod cell walls are homogeneous in size and average 114 to 135 hexosamines per chain.     

Heterologous expression and characterization of Choline Oxidase from the soil bacterium Arthrobacter nicotianae

In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15–70 °C). Specific activities were determined toward choline chloride (4.70 ± 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 ± 0.45 × 10^–2 U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 ± 0.12 × 10^–2 U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K _M = 1.51 ± 0.09 mM and V _max = 42.73 ± 0.42 mU/min for choline chloride and K _M = 4.77 ± 0.76 mM and V _max = 48.40 ± 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.     

Biosynthetic features and properties of xylose isomerases from Arthrobacter nicotianae, Escherichia coli, and Erwinia carotovota subsp. atroseptica

The characteristics of xylose isomerase biosynthesis in the bacteria Arthrobacter nicotianae BIM B-5, Erwinia carotovota subsp atroseptica jn42xylA, and Escherichia coli HB101xylA have been studied. The bacteria formed the enzyme constitutively. Out of the carbon sources studied, D-glucose and D-xylose were most favorable for the biosynthesis of xylose isomerase in E. carotovota subsp atroseptica, but the least appropriate in terms of the enzyme production efficiency in E. coli. Minimum and maximum levels of xylose isomerase formation in A. nicotianae were noted, respectively, during D-xylose and sucrose utilization. An addition to the nutrient medium of 0.1-1.5% D-glucose (together with D-xylose) did not affect the enzyme synthesis in A. nicotianae, but suppressed it in Erwinia carotovota subsp atroseptica (by 7% at the highest concentration) and Escherichia coli (by 63 and 75% at concentrations of 0.1 and 1.0%, respectively). The enzyme proteins produced by the bacteria exhibited the same substrate specificity and electrophoretic mobility (PAGE) as xylose isomerase A. nicotianae, although insignificant differences in the major physicochemical properties were noted.     

Biosynthetic features and properties of xylose isomerases from Arthrobacter nicotianae, Escherichia coli, and Erwinia carotovora subsp. atroseptica

The characteristics of xylose isomerase biosynthesis in the bacteria Arthrobacter nicotianae BIM B-5, Erwinia carotovora subsp atroseptica jn42xylA, and Escherichia coli HB101xylA have been studied. The bacteria produced the enzyme constitutively. Out of the carbon sources studied, D-glucose and D-xylose were most favorable for the biosynthesis of xylose isomerase in E. carotovora subsp. atroseptica, but the least appropriate in terms of the enzyme production efficiency in E. coli. Minimum and maximum levels of xylose isomerase formation in A. nicotianae were noted, respectively, during D-xylose and sucrose utilization. An addition to the D-xylose-containing nutrient medium of 0.1–1.5% D-glucose did not affect the enzyme synthesis in A. nicotianae, but suppressed it in Erwinia carotovora subsp. atroseptica (by 7% at the highest concentration) and Escherichia coli (by 63 and 75% at concentrations of 0.1 and 1.0%, respectively). The enzyme proteins produced by the bacteria exhibited the same substrate specificity and electrophoretic mobility (PAGE) as xylose isomerase A. nicotianae, although insignificant differences in the major physicochemical properties were noted.     

Mechanism of reversible glycine cleavage reaction in Arthrobacter globiformis. Function of lipoic acid in the cleavage and synthesis of blycine.

The physical and chemical characterization of horse serum butyrylcholinesterase has been extended. The results show that the enzyme is a glycoprotein containing about 20% carbohydrate by weight. Mannose, glucosamine, galactose, and sialic acid are the sugar residues found. The extinction coefficient of butyrylcholinesterase, E_1cm^1% at 280 nm, was found to be 15.2 ± 0.3 by dry weight determination. The molecular weight of the protein in dilute phosphate buffer was determined to be (31.7 ± 1.2) × 10⁴ by high speed equilibrium sedimentation with a redetermined partial specific volume of 0.723 ± 0.003 ml/g. Subunit molecular weights for the dissociated protein were found to be (7.9 ± 0.4) × 10⁴ and (8.1 ± 0.1) × 10⁴, respectively, in guanidine hydrochloride and in a solution at pH 11.8. The subunit molecular weight was also estimated to be (8.8 ± 0.2) × 10⁴ by analytical sodium dodecyl sulfate-gel electrophoresis. This apparently higher subunit molecular weight from dodecyl sulfate gels is expected for glycoproteins containing significant amounts of carbohydrate. No free sulfhydryl group was detected, even though there are six half-cystines in each subunit. Therefore, it seems likely that there are three pairs of disulfide bonds per subunit. The available data indicate that native butyrylcholinesterase is a tetrameric glycoprotein consisting of subunits of equal molecular weight.