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PubMed2003

Cold adaptation of a psychrophilic chitinase: a mutagenesis study.

The gene encoding chitinase ArChiB from the Antarctic Arthrobacter sp. TAD20 has been expressed in Escherichia coli and the recombinant enzyme purified to homogeneity. In an effort to engineer cold-adapted biocatalysts through rational redesign to operate at elevated temperatures, we performed several mutations aiming to increase the rigidity of the molecular edifice of the selected psychrophilic chitinase. The mutations were designed on the basis of a homology-based three-dimensional model of the enzyme, and included an attempt to introduce a salt bridge (mutant N198K) and replacements of selected Gly residues by either Pro (mutants G93P, G254P) or Gln (G406Q). Mutant N198K resulted in a more stable protein (DeltaTm = 0.6 degrees C). Mutant G93P exhibited a DeltaTm of 1.2 degrees C, while mutants G254P and G406Q exhibited decreased stability. We conclude that the effect of mutating Gly residues on enzyme stability is rather complex and can only be understood in the context of the structural environment. Kinetic and spectroscopic analysis of these enzyme variants revealed that the kinetic parameters kcat and Km have been significantly modified.

Adaptation, Biological↗

PubMed1982

A survey of the structures of mycolic acids in Corynebacterium and related taxa.

Acid methanolysates of 104 strains of coryneform and related bacteria were examined for mycolic acids by thin-layer chromatography and mass spectrometry. The mycolic acids of Corynebacterium sensu stricto strains were not uniform in size and structure, and considerable variation existed within the approximate limiting range of C22 to C36. Two species, Corynebacterium bovis and 'Corynebacterium mycetoides', were particularly distinctive; the former had exceptionally low molecular weight mycolic acids (C22 to C32), whereas the latter contained major amounts of mycolates with a side-chain possessing an odd number of carbon atoms. Caseobacter polymorphus contained mycolic acids of a similar size (C30 to C36) to those of true corynebacteria. The mycolates of organisms assigned to the genus Rhodococcus were generally larger (C30 to C56), although a clear distinction between two corynebacteria and rhodococci cannot presently be made by analysis of mycolic acids alone. Strains labelled 'Arthrobacter roseoparaffinus', Corynebacterium equi and Corynebacterium hoagii contained mycolic esters with molecular weights 'intermediate' in size between those of true corynebacteria and rhodococci. The mycolic acid data correlate well with other major trends in coryneform taxonomy and support earlier suggestions that they are of value in the classification of the group.

Arthrobacter↗

PubMed1990

Characterization of phi GA1, an inducible phage particle from Brevibacterium flavum.

Eighteen related strains of Arthrobacter, Brevibacterium and Corynebacterium were used as indicator strains in an attempt to isolate corynephages from a large number of soils and from waste-water samples. Although no phages capable of producing plaques were isolated, one of the indicator strains used, Brevibacterium flavum ATCC 14067, was lysogenic for the inducible phage phi GA1. This phage was not observed to form plaques on any of the strains tested. phi GA1 is of B1-morphotype with a linear double-stranded DNA genome of 48.1 kb with cohesive ends; a restriction map is presented.

Arthrobacter↗

PubMed1976

Thin-layer chromatographic analysis of mycolic acid and other long-chain components in whole-organism methanolysates of coryneform and related taxa.

Acid methanolysates of strains representing 58 coryneform taxa were examined for mycolic acids and other long-chain constituents by thin-layer chromatography. Mycolic esters were detected in the methanolysates of true corynebacteria but not in those from plant pathogenic bacteria, Corynebacterium haemolyticum, Corynebacterium pyogenes or from representatives of the genera Arthrobacter, Cellulomonas, Curtobacterium, Kurthia or Oerskovia. Thin-layer chromatography of whole-organism methanolysates provides a simple method for distinguishing true corynebacteria from coryneforms which do not contain mycolic acids, and from nocardiae and mycobacteria which produce mycolic acids of different mobility. At present the mycolic esters of true corynebacteria cannot be clearly separated from those of some rhodochrous strains.

Actinomycetales↗

PubMed1995

Selective recovery of Streptosporangium fragile from soil by indirect immunomagnetic capture.

A polyclonal antibody raised to Streptosporangium fragile spores reacted strongly and specifically with the immunizing strain and to a number of related species of Streptosporangium, as determined by dot immunoblotting. An indirect immunomagnetic capture method was developed for the recovery of the target organism from sterile and non-sterile soil, using sheep anti-rabbit M-280 Dynabeads. The effects of different soil blocking agents, antibody labelling concentrations and spore/Dynabead capture times on the recovery of S. fragile spores were investigated. Pre-blocking of antibody binding sites within the soil, with either 2% partially hydrolysed gelatin or 10% skimmed milk, was essential prior to immunomagnetic capture. Increasing the capture time from 15 to 60 min did not affect spore recovery; however, a 10-fold decline in the magnetic bead concentration did result in a significantly lower recovery of spores from soil. S. fragile was selectively enriched (1:190-fold) when present as a mixed population with Arthrobacter oxydans in sterile soil. The indirect immunomagnetic capture method was used to selectively recover S. fragile spores seeded into non-sterile soil, although some background binding of non-target bacteria was noted. The target was successfully recovered from a sterile soil microcosm after 14 d incubation and the capture rate was increased by the inclusion of an initial soil dispersion and biomass concentration procedure, using the ion-exchange resin Chelex 100.

Actinomycetales↗

PubMed2004

Stimulation of phagocytosis of influenza virus-infected cells through surface desialylation of macrophages by viral neuraminidase.

Cells infected with influenza A virus undergo apoptosis and become susceptible to phosphatidylserine-mediated phagocytosis by macrophages. This study was undertaken to elucidate the mechanism underlying our previous finding that the activity of viral neuraminidase (NA) is required for efficient phagocytosis. Treatment of macrophages, not influenza virus-infected cells, with Arthrobacter ureafaciens NA or virus-infected cells expressing viral NA augmented the level of phagocytosis of virus-infected cells but not of latex beads or cells undergoing Fas-induced apoptosis. Oligosaccharides, including sialyllactose, bound to influenza virus-infected cells and inhibited phagocytosis by macrophages. These results indicate that surface desialylation of macrophages by influenza virus NA modulates the mode of association between macrophages and target virus-infected cells and stimulates phosphatidylserine-mediated phagocytosis.

Animals↗

PubMed1995

A phylogenetic analysis of micro-organisms isolated from subsurface environments.

Three methods were used to provide information on the identity and phylogenetic relatedness of 19 aerobic, chemoheterotrophic bacteria isolated from topsoil and deep subsurface sediments at a site in South Carolina. These methods were (i) analysis of selected physiological traits, (ii) restriction endonuclease analysis (REA) of genomic DNA, and (iii) analysis of 16S ribosomal RNA sequences. When the 16S rRNA sequences were compared with those for 12 standard strains, two topsoil isolates and six subsurface strains formed a tight group with the high-G+C Gram-positive bacteria and appeared to be most closely related to Arthrobacter globiformis--a coryneform-actinomycete bacterium with unusually effective survival capabilities. The rest of the subsurface isolates were scattered among the standard strains from the Proteobacteria-including the pseudomonads and Agrobacterium tumefaciens--or the low-G+C Gram-positive bacteria.

Agrobacterium tumefaciens↗

PubMed2004

Genetic engineering of glycinebetaine synthesis in tomato protects seeds, plants, and flowers from chilling damage.

Tomato (Lycopersicon esculentum Mill.) plants, which normally do not accumulate glycinebetaine (GB), are susceptible to chilling stress. Exposure to temperatures below 10 degrees C causes various injuries and greatly decreases fruit set in most cultivars. We have transformed tomato (cv. Moneymaker) with a chloroplast-targeted codA gene of Arthrobacter globiformis, which encodes choline oxidase to catalyze the conversion of choline to GB. These transgenic plants express codA and synthesize choline oxidase, while accumulating GB in their leaves and reproductive organs up to 0.3 and 1.2 micromol g(-1) fresh weight (FW), respectively. Their chloroplasts contain up to 86% of total leaf GB. Over various developmental phases, from seed germination to fruit production, these GB-accumulating plants are more tolerant of chilling stress than their wild-type counterparts. During reproduction, they yield, on average, 10-30% more fruit following chilling stress. Endogenous GB contents as low as 0.1 micromol g(-1) FW are apparently sufficient to confer high levels of tolerance in tomato plants, as achieved via transformation with the codA gene. Exogenous application of either GB or H2O2 improves both chilling and oxidative tolerance concomitant with enhanced catalase activity. These moderately increased levels of H2O2 in codA transgenic plants, as a byproduct of choline oxidase-catalyzed GB synthesis, might activate the H2O2-inducible protective mechanism, resulting in improved chilling and oxidative tolerances in GB-accumulating codA transgenic plants. Thus, introducing the biosynthetic pathway of GB into tomato through metabolic engineering is an effective strategy for improving chilling tolerance.

Alcohol Oxidoreductases↗

PubMed2006

Allergenicity assessment of transgenic mustard (Brassica juncea) expressing bacterial codA gene.

BACKGROUND: Assessing the allergenicity and toxicity of genetically modified (GM) crops is essential before they become a regular part of our food supply. The present study aimed to assess the allergenicity of Brassica juncea (mustard) expressing choline oxidase (codA) gene from Arthrobacter globiformis that provides resistance against abiotic stresses. METHODS: SDAP, Farrp, and Swiss-Prot databases were used to study allergenicity of choline oxidase. Digestibility of choline oxidase was assessed in simulated gastric fluid (SGF). Specific immunoglobulin E (IgE) reactivity of native and GM mustard was compared by using enzyme-linked immunosorbent assay (ELISA) and skin tests in respiratory-allergic patients. Allergenicity of GM and native mustard proteins was compared in Balb/c mice. RESULTS: Choline oxidase showed no significant homology with allergenic proteins in SDAP and Farrp databases. Cross-reactive epitope search showed a stretch similar to Hev b 6 having some antigenic properties. Purified choline oxidase showed complete degradation with SGF. Skin prick test of native and GM mustard extract on respiratory allergic patients showed significant correlation (P < 0.05). ELISA with 96 patients' sera showed comparable IgE reactivity. Balb/c mice immunized with native and GM mustard proteins showed low IgE response. Presensitized mice on intravenous challenge with Brassica extract showed no anaphylactic symptoms unlike ovalbumin (OVA) sensitization that showed anaphylactic reaction in mice. Lung histology of OVA-sensitized mice showed narrowing of airway and large eosinophilic infiltration, whereas native and GM Brassica extract showed normal airway. CONCLUSION: Genetically modified mustard with the codA gene possessed allergenicity similar to that of native mustard and no enhancement of IgE binding was observed due to genetic manipulation.

Adolescent↗

PubMed1976

Restriction-enzyme-cleavage maps of bacteriophage M13. Existence of an intergenic region on the M13 genome.

Replicative form DNA of bacteriophage M13 was cleaved into specific fragments by an endonuclease isolated from Hemophilus aegyptius (endoR.HaeII) and an endonuclease from Arthrobacter luteus (endoR.AluI). The fragments were ordered as to construct a circular map of the phage M13 genome by: (a) using each fragment as a primer for the synthesis in vitro of its respective neighbour and (b) digesting the isolated fragments with the Hemophilus aegyptius enzyme endoR.HaeII or the Hemophilus aphirophilus enzyme endoR.HapII and subsequent analysis of the overlapping sets of fragments. The resulting physical map was correlated with the M13 genetic map by marker rescue experiments with amber mutant phage DNAs and purified wild-type fragments. From the results of these analyses it has been concluded that gene II and gene V are contiguous on the genetic map. Evidence is provided that there is an internal start of RNA synthesis within the C-terminal region of gene II which then ultimately leads to the synthesis of X protein. Furthermore, we conclude that there is an intergenic space of considerable length (450-500 base pairs) which is located between gene II and gene IV on the M13 genome. The function of this intergenic region as the origin site for phage DNA replication is discussed.

Arthrobacter↗

PubMed1977

Comparative analysis of three guinea pig satellite DNA's by restriction nucleases.

The structures of guinea pig satellite DNAs I, II, and III have been analyzed by digestion with seven restriction nucleases. From the cleavage patterns it is obvious that the long-range periodicities in these three satellites differ rather characteristically Satellite I is fairly resistant to six nucleases and gives only a number of weak discrete bands which do not show a simple regularity. By the restriction nuclease from Arthrobacter luteus, however, it is cleaved extensively and yields very heterogeneous breakdown products. This is consistent with the high extent of divergence previously found for this satellite, e. g. by sequence analysis. Satellite II is almost completely resistant to all nucleases, indicative of a high degree of sequence homogeneity of this satellite. Satellite III is completely broken by the restriction nuclease from Bacillus subtilis into fragments which form a novel, highly regular series of bands in gel electrophoresis. The patterns show that the satellite is composed of tandem repeats ofapproximately 215 nucleotide pairs length, each repeat unit containing two cleavage sites for this nuclease. The data are consistent with the assumption that 30--40% of all cleavage sites have been eliminated by a random process. Satellite III DNA yields weak degradation patterns of the same periodicity with a number of other restriction nucleases. Cleavage sites for these nuclease are clustered on separatesmall segments of the satellite DNA. In this respect, the satellite is similar to others, notably the mouse satellite DNA. The three guinea pig satellites are examples of more general types of satellite structures also found in othe organisms. Similarities and differences to other satellites are discussed with special consideration to theories on the evolution of this class of DNA.

Animals↗

PubMed1978

FAD is covalently attached to peptidyl-tRNA during cell-free synthesis of 6-hydroxy-D-nicotine oxidase.

The process, by which FAD is attached covalently to the 6-hydroxy-D-nicotine oxidase apoprotein in D-nicotine-induced cells of Arthrobacter oxidans was studied in vitro. [3H]Adenine-labelled FAD prepared biosynthetically in Clostridium kluyveri was incorporated into the 6-hydroxy-D-nicotine oxidase molecule during cell-free translation. FAD rather than FMN or riboflavin was thus shown to be the flavin derivative transferred to the polypeptide chain. After short-term protein synthesis on ribosomes from induced A. oxidans cells in the presence of an Escherichia coli MRE 600 supernatant fraction and [adenine-2-3H]FAD, THE PEPTIDYL-TRNA fraction was separated from completed polypeptides. Labelled FAD was found to be covalently attached to the tRNA-bound polypeptides. Cleavage of the tRNA-peptide bond released labelled polypeptides the largest of which migrated as authentic 6-hydroxy-D-nicotine oxidase during dodecylsulfate/polyacrylamide gel electrophoresis. These results strongly suggest that FAD is incorporated into the nascent polypeptide chains of 6-hydroxy-D-nicotine oxidase during ribosomal translation.

Adenine↗

PubMed1979

Induction of the phosphoenolpyruvate: hexose phosphotransferase system associated with relative anaerobiosis in an obligate aerobe.

Arthrobacter pyridinolis possesses alternative transport systems for D-fructose: a respiration-coupled transport system whereby D-fructose transport occurs with concomitant oxidation of L-malate, and a phosphoenolpyruvate: D-fructose phosphotransferase system. Studies of D-fructose uptake by whole cells in the presence and absence of cyanide demonstrate that respiration-coupled transport is used almost exclusively during the first half of logarithmic growth, after which it accounts for only 15-20% of D-fructose uptake. Phosphotransferase levels are low during log phase, peak during late log, and then slowly decline. In a mutant of A. pyridinolis which requires delta-aminolevulinic acid for growth, the growth rate, cell cytochrome content, and activity of the respiration-coupled transport system increased with increasing concentrations of delta-aminolevulinic acid up to 50 microgram/ml. By contrast, phosphotransferase activity was highest in cells grown on limiting delta-aminolevulinic acid. L-Malate, which stimulates respiration-coupled transport, repressed the phosphotransferase system. The respiratory activity and the ability to release CO2 from internalized d-fructose was consistently low in D-fructose-grown cells. A cyanide-resistant cytochrome, tentatively identified as cytochrome d, appeared in the late exponential phase of growth. Isocitrate lyase activity, required for aerobic growth of this organism, declined markedly during the late exponential phase. Thus the phosphotransferase system is maximally induced, in this obligate aerobe, under conditions of relative anaerobiosis during which metabolism is primarily fermentative.

Aerobiosis↗

PubMed1980

Cell-free synthesis of a flavoprotein containing the 8 alpha-(N3-histidyl)-riboflavin linkage.

6-Hydroxy-D-nicotine oxidase is an inducible flavorprotein of Arthrobacter oxidans in which one mole of FAD is bound covalently to the polypeptide chain. During cell-free translation of polysomes from nicotine-induced A. oxidans cells in the presence of an Escherichia coli (MRE 600) supernatant fraction, labelled FAD, leucine and histidine were incorporated into 6-hydroxy-D-nicotine oxidase in the same ratio found in the enzyme isolated from whole cells. This indicates that one mole FAD is covalently attached per mol of 6-hydroxy-D-nicotine oxidase synthesized in vitro. In the native enzyme the coenzyme molecule is bound via its 8 alpha-methyl group to the N-3 atom of a histidyl residue. From the translation products an aminoacyl-riboflavin was isolated and its identity with synthetic 8 alpha-(N3-histidyl)-riboflavin was shown.

Arthrobacter↗

PubMed1986

Studies in vitro on the flavinylation of 6-hydroxy-D-nicotine oxidase.

The gene of 6-hydroxy-D-nicotine oxidase (6-HDNO), a flavoenzyme from Arthrobacter oxidans with covalently bound FAD, was expressed with the aid of an expression vector in a cell-free coupled transcription-translation system derived from Escherichia coli MZ9. Ultraviolet irradiation of the E. coli extract did not affect synthesis of the 6-HDNO polypeptide nor total protein synthesis but enzymatic 6-HDNO activity could not be detected. Addition of FAD to the irradiated cell extract restored the capability of the transcription-translation assays to synthesize enzymatically active 6-HDNO. However, enzymatic activity could not be restored on addition of FAD plus cell-free extract to the ultraviolet-inactivated assays after completion of apo-6-HDNO synthesis (60 min) nor to immunoprecipitates thereof. Under similar conditions, addition of [14C]FAD did not increase the protein-bound radioactivity. These results indicate that under conditions of limited FAD supply in the in vitro system a flavinless apo-6-HDNO-polypeptide was synthesized. It was, however, not possible to bind the cofactor to the completed polypeptide chain. These findings argue for a cotranslational cofactor binding.

Arthrobacter↗

PubMed1987

Covalent flavinylation of 6-hydroxy-D-nicotine oxidase analyzed by partial deletions of the gene.

The expression of the enzymatically active 6-hydroxy-D-nicotine oxidase (6-HDNO) from Arthrobacter oxidans requires the covalent attachment of FAD to the polypeptide chain. How this modification takes place and at what time during the synthesis of the polypeptide is not known. We investigated the possibility of cotranslational flavinylation by generating various deletions of the 6-HDNO gene carried on appropriate plasmid vectors. The polypeptides expressed from these plasmids were analyzed for their ability to incorporate [14C]FAD covalently in an Escherichia coli-derived coupled transcription/translation system. The data show that removal of approximately 40% from the carboxy-terminal part of the 6-HDNO polypeptide did not inhibit the covalent flavinylation of the truncated protein. A fusion protein, consisting of the truncated 6-HDNO polypeptide and the beta-lactamase of pBR322, was also covalently flavinylated. The amino acid sequence surrounding the histidine residue, assumed to bind FAD, was shown to be situated approximately 70 amino acid residues from the amino-terminal end of the 6-HDNO polypeptide. Removal of the first 30 amino acids did not abolish covalent flavinylation. Flavinylation could no longer be detected, however, if a short amino acid sequence, consisting of seven residues, replaced the amino acid sequence upstream of the histidine. These findings prove, in our opinion, that cotranslational flavinylation takes place in the synthesis of 6-HDNO.

Amino Acid Sequence↗

PubMed2005

Desulphurization of dibenzothiophene and diesel oils by bacteria.

AIMS: To study the desulphurization of dibenzothiophene (DBT), a recalcitrant thiophenic component of fossil fuels, by two bacteria namely Rhodococcus sp. and Arthrobacter sulfureus isolated from oil-contaminated soil/sludge in order to use them for reducing the sulphur content of diesel oil in compliance with environmental regulations. METHODS AND RESULTS: The desulphurization pathway of DBT by the two bacteria was determined by gas chromatography (GC) and GC-mass spectrometry. Both organisms were found to produce 2-hydroxy biphenyl (2-HBP), the desulphurized product of DBT. Sulphur contents of culture supernatants of Rhodococcus sp. and A. sulfureus grown with DBT as sole sulphur source were analysed by X-ray fluorescence indicating sulphur levels of 8 and 10 ppm, respectively, as compared with 27 ppm in control. In order to study desulphurization of diesel oils obtained from an oil refinery, resting cell studies were carried out which showed a decrease of about 50% in sulphur content of the oil obtained from the hydrodesulphurization (HDS) unit of the refinery. CONCLUSIONS: Rhodococcus sp. and A. sulfureus selectively remove sulphur from DBT to form 2-HBP. Application of these bacteria for desulphurization of diesel showed promising potential for decreasing the sulphur content of diesel oil. SIGNIFICANCE AND IMPACT OF THE STUDY: The process of microbial desulphurization described herein can be used for significantly reducing the sulphur content of oil, particularly, after the process of HDS which would help in meeting the regulatory standards for sulphur level in diesel oil.

Arthrobacter↗

PubMed2006

Bacterial community structure of a pesticide-contaminated site and assessment of changes induced in community structure during bioremediation.

The introduction of culture-independent molecular screening techniques, especially based on 16S rRNA gene sequences, has allowed microbiologists to examine a facet of microbial diversity not necessarily reflected by the results of culturing studies. The bacterial community structure was studied for a pesticide-contaminated site that was subsequently remediated using an efficient degradative strain Arthrobacter protophormiae RKJ100. The efficiency of the bioremediation process was assessed by monitoring the depletion of the pollutant, and the effect of addition of an exogenous strain on the existing soil community structure was determined using molecular techniques. The 16S rRNA gene pool amplified from the soil metagenome was cloned and restriction fragment length polymorphism studies revealed 46 different phylotypes on the basis of similar banding patterns. Sequencing of representative clones of each phylotype showed that the community structure of the pesticide-contaminated soil was mainly constituted by Proteobacteria and Actinomycetes. Terminal restriction fragment length polymorphism analysis showed only nonsignificant changes in community structure during the process of bioremediation. Immobilized cells of strain RKJ100 enhanced pollutant degradation but seemed to have no detectable effects on the existing bacterial community structure.

Arthrobacter↗