Al.ca.li' ge.nes . Arabic al the; Arabic n. galiy the ash of saltwort; French n. alcali alkali; Gr. v. gennaio to produce; M.L. masc. n. Alcaligenes alkali‐producing (bacteria). Proteobacteria / Betaproteobacteria / Burkholderiales / Alcaligenaceae / Alcaligenes Rods or coccobacilli , 0.5–1.2 × 1.0–3.0 µm, usually occurring singly. Resting stages not known. Gram negative . Motile with one to nine peritrichous flagella. Obligately aerobic , possessing a strictly respiratory type of metabolism with oxygen as the terminal electron acceptor. Some strains are capable of anaerobic respiration in the presence of nitrate or nitrite. Optimal growth temperature: 20–37°C. Colonies on nutrient agar are nonpigmented . Oxidase positive . Catalase positive . Indole not produced. Cellulose, esculin, gelatin, and DNA usually not hydrolyzed. Chemoorganotrophic , using a variety of organic acids and amino acids as carbon sources . Alkali produced from several organic salts and amides. Carbohydrates usually not utilized. Characteristic fatty acids are C 17 : 0 cyclo , C 16 : 0 , C 14 : 0 3OH , C 16 : 1 , and C 12 : 0 2OH . Ubiquinone Q‐8 . Polyamine patterns with the predominant compound putrescine and the unusual diamine 2‐hydroxyputrescine . Isolated from water, soil, and clinical specimens such as blood, spinal fluid, pleural fluid, peritoneal fluid, pus, urine, stools, and swabs of eyes, ears, and pharynxes. Frequently found in unsterilized distilled water and in chlorhexidine solutions in hospitals. Occasionally causing opportunistic infections in humans. The mol % G + C of the DNA is : 56–60. Type species : Alcaligenes faecalis Castellani and Chalmers 1919, 936 AL ( Achromobacter arsenoxydans‐tres Turner 1954, 475; “ Pseudomonas odorans ” (Málek and Kazdová‐Košiskova 1946; Málek, Radochová and Lysenko 1963, 353; “ Alcaligenes odorans var. viridans” Mitchell and Clarke 1965, 347.

A new cyclic hexapeptide, baceridin (1), was isolated from the culture medium of a plant-associated Bacillus strain. The structure of 1 was elucidated by HR-HPLC-MS and 1D and 2D NMR experiments and confirmed by ESI MS/MS sequence analysis of the corresponding linear hexapeptide 2. The absolute configurations of the amino acid residues were determined after derivatization by GC-MS and Marfey's method. The cyclopeptide 1 consists partially of nonribosomal-derived D- and allo-D-configured amino acids. The order of the D- and L-leucine residues within the sequence cyclo(-L-Trp-D-Ala-D-allo-Ile-L-Val-D-Leu-L-Leu-) was assigned by total synthesis of the two possible stereoisomers. Baceridin (1) was tested for antimicrobial and cytotoxic activity and displayed moderate cytotoxicity (1-2 gmL(-1)) as well as weak activity against Staphylococcus aureus. However, it was identified to be a proteasome inhibitor that inhibits cell cycle progression and induces apoptosis in tumor cells by a p53-independent pathway.

Ribonucleotide reduction, the unique step in the pathway to DNA synthesis, is catalyzed by enzymes via radical‐dependent redox chemistry involving an array of diverse metallocofactors. The nucleotide reduction gene (nrdF) encoding the metallocofactor containing small subunit (R2F) of the Corynebacterium ammoniagenes ribonucleotide reductase was reintroduced into strain C. ammoniagenes ATCC 6872. Efficient homologous expression from plasmid pOCA2 using the tac‐promotor enabled purification of R2F to homogeneity. The chromatographic protocol provided native R2F with a high ratio of manganese to iron (30 : 1), high activity (69 μmol 2′‐deoxyribonucleotide·mg−1·min−1) and distinct absorption at 408 nm, characteristic of a tyrosyl radical (Y˙), which is sensitive to the radical scavenger hydroxyurea. A novel enzyme assay revealed the direct involvement of Y˙ in ribonucleotide reduction because 0.2 nmol 2′‐deoxyribonucleotide was formed, driven by 0.4 nmol Y˙ located on R2F. X‐band electron paramagnetic resonance spectroscopy demonstrated a tyrosyl radical at an effective g‐value of 2.004. Temperature dependent X/Q‐band EPR studies revealed that this radical is coupled to a metallocofactor. Similarities of the native C. ammoniagenes ribonucleotide reductase to the in vitro activated Escherichia coli class Ib enzyme containing a dimanganese(III)‐tyrosyl metallocofactor are discussed.

The X-ray crystallographic structure of the native R2F subunit of the ribonucleotide reductase (RNR) of Corynebacterium ammoniagenes ATCC 6872 is reported, with a resolution of 1.36 A. The metal site contains an oxo/hydroxo-bridged manganese dimer, located near a tyrosine residue (Y115). The coordination of the manganese dimer and its distance to a nearby tyrosine residue resemble the di-iron metalloradical cofactor of class I RNR from Escherichia coli . Multifrequency EPR measurements of the highly active C. ammoniagenes R2F subunit show that the metal site contains a ferromagnetically exchange-coupled Mn(III)Mn(III) dimer weakly coupled to a tyrosyl radical. A mechanism for the metalloradical cofactor (Mn(III)Mn(III)Y(*)) generation is proposed. H(2)O(2) (HO(2)(-)) instead of O(2) is hypothesized as physiological oxidant for the Mn dimer which in turn oxidizes the tyrosine Y115. Changes in the ligand sphere of both manganese ions during metalloradical generation direct the complex formation of this cofactor, disfavoring alternate reaction pathways such as H(2)O(2) dismutation, as observed for manganese catalase, a structural analogue of the R2F metal site. The presented results demonstrate the importance of manganese for radical formation in this RNR and confirm the assignment of this enzyme to class Ib.

Bacterial shoot blight of pear in Japan (BSBP) is caused by Erwinia strains which were formerly associated with the species Erwinia amylovora, the causative agent of fire blight. The description of Erwinia pyrifoliae as a pear pathogen in Korea renewed a possible connection of the pear pathogens in both countries.Nucleotide sequence analysis of the 16S rRNA, the house keeping genes gpd and recA, as well as DNA-DNA hybridization kinetics and microbiological assays place the pear pathogens from Japan into the species E. pyrifoliae described as the causative agent of Asian pear blight in Korea.Erwinia pyrifoliae strains from Korea and the pear pathogenic Erwinia strains from Japan belong taxonomically into the same species, but show slight divergences in nucleotide sequences used for classification. The allocation is not only supported by microbiological properties, but also by a host range restricted to pear observed before by others.The data suggest that the BSBP disease observed at the island of Hokkaido was not fire blight and unify BSBP in Japan with the pear pathogenic species E. pyrifoliae from Korea.

Ribonucleotide reduction, the unique step in DNA-precursor biosynthesis, involves radical-dependent redox chemistry and diverse metallo-cofactors. The metallo-cofactor (R2F) encoded by the nrdF (nucleotide reduction) gene in Corynebacterium ammoniagenes ATCC 6872 was isolated after homologous expression and a new crystal form of ribonucleotide reductase R2F was obtained. R2F was crystallized at 277 K using the vapour-diffusion method with PEG as the precipitating agent. A data set was collected to 1.36 A resolution from a single crystal at 100 K using synchrotron radiation. The crystal belonged to space group C2, with unit-cell parameters a = 96.21, b = 87.68, c = 83.25 A, beta = 99.29 degrees. The crystal contained two molecules per asymmetric unit, with a Matthews coefficient (V(M)) of 2.69 A(3) Da(-1); the solvent content was estimated to be 54.3%. X-ray fluorescence spectroscopy and MAD diffraction data indicated the presence of manganese in the molecule and the absence of iron.

Ribonucleotide reductases (RNR; EC 1.17.4.1) provide the 2′-deoxyribonucleotides for DNA replication of proliferating cells by a uniform radical mechanism using diverse metals. The native metallo-cofactor of the Corynebacterium glutamicum RNR contains manganese and is sensitive to EDTA and radical scavengers. Hybrid holoenzymes, capable of ribonucleotide reduction, were composed of the small manganese-containing (R2F) and the large catalytic subunit (R1E) from either of the two corynebacterial RNRs. A synthetic peptide deduced from the C-terminal region of the nrdF gene inhibited the C. glutamicum-RNR non-competitively and cross-reacted with the C. ammoniagenes-RNR. The C. glutamicum-R2F has a saturable organic radical signal at g=2.005 detected by electron paramagnetic resonance (EPR) spectroscopy and shows a distinct absorption at 408 nm indicative of a tyrosyl-like organic radical (Y·). Quantification of the metal content revealed 0.06 mol Fe but 0.8 mol Mn per mol R2F-monomer and would thus assign two manganese atoms bound to the dimeric metallo-cofactor, while a distinct enzymatic activity (32 µmol×mg−1×min−1) was observed in the biochemical complementation assay. Divergence of the C. glutamicum-RNR studied here from the prototypical Salmonella typhimurium class 1b enzyme and the Chlamydia trachomatis class Ic enzyme is discussed below.

A collection of highly nickel and cobalt-resistant enterobacteria were isolated from the Punta Gorda serpentine deposit (Moa, Cuba). The most nickel and cobalt resistant strain (termed C- 1) was assigned to Serratia marcescens by 16S rRNA analysis and DNA/DNA hybridization and the molecular mechanisms underlying its inducible cobalt and nickel resistance was investigated. Genes involved in metal resistance were identified by transposon mutagenesis followed by selection for Co- and Ni-sensitive derivatives. The transposon insertion causing the highest decrease in metal resistance was located in the ncrABC determinant. The three ORFs (ncrA, ncrB and ncrC) were cloned in E. coli. The predicted NcrA product was an NreB ortholog of the major facilitator protein superfamily and was central for Co/Ni resistance in S. marcescens strain C-1. NcrA also mediated metal resistance in E. coli and caused decreased accumulation of Co and Ni in this heterologous host. NcrB may be a regulatory protein. NcrC was a protein of the Ni–Co transport (NiCoT) protein family and necessary for full metal resistance in E. coli, but only when NcrA was also present. Without NcrA, NcrC caused a slight decrease in metal resistance and mediated increased accumulation of Ni and Co. As the cytoplasmic metal concentration can be assumed to be the result of a flow equilibrium of uptake and efflux processes, this interplay between metal uptake system NcrC and metal efflux system NcrA may contribute to nickel and cobalt resistance in this bacterium.

Strains L10(T), L108 and CIP I-2052 were originally obtained from methyl tert-butyl ether (MTBE)-contaminated groundwater and from a wastewater treatment plant, respectively. All share the ability to grow on tert-butanol, an intermediate of MTBE degradation. Cells are strictly aerobic, motile by a polar flagellum and exhibit strong pili formation. Poly beta-hydroxybutyrate (PHB) granules are formed. The DNA G+C content is 69-70.5 mol% and the main ubiquinone is Q-8. The major cellular fatty acids are 16 : 1 cis-9 and 16 : 0 and the only hydroxy fatty acid is 10 : 0 3-OH. The major phospholipids are phosphatidylethanolamine (PE) 16 : 1/16 : 1 and phosphatidylglycerol 16 : 0/16 : 1. A significant amount of PE 17 : 0/16 : 1 is present. The 16S rRNA gene sequences of these strains are almost identical and form a separate line of descent in the Rubrivivax-Roseateles-Leptothrix-Ideonella-Aquabacterium branch of the Betaproteobacteria with 97 % similarity to 16S rRNA genes of the type strains of Rubrivivax gelatinosus, Leptothrix mobilis and Ideonella dechloratans. However, physiological properties, DNA-DNA relatedness values and the phospholipid and cellular fatty acid profiles distinguish the novel isolates from the three closely related genera. Therefore, it is concluded that strains L10(T), L108 and CIP I-2052 represent a new genus and novel species for which the name Aquincola tertiaricarbonis gen. nov., sp. nov., is proposed. The type strain is strain L10(T) (=DSM 18512(T)=CIP 109243(T)).

The general hypothesis of enhanced membranepermeability as a cause of increased production of primary and secondary metabolites (the "leak model") was disproved for the nucleotide (NAD) fermentation with Brevibacterium ammoniagenesATCC6872, as manganese deficient producer cells displayed a high membrane potential, like non-producing cells supplied with 10 jim Mn2+. However, the protein composition of the membrane was affected by a manganese deficiency. A manganese deficiency-like protein pattern of the membrane could be induced, in the presence of 10 jim Mn2+, by either inhibition of DNAprecursor biosynthesis (ribonucleotide reduction) with 20mM hydroxyurea (HU) or inhibition of DNAreplication with 30/*M mitornvein C.

Bacteria were isolated from flowers and bark of apple and pear trees at three places in Australia. In Victoria, Tasmania and Queensland, strains with white colonies on nutrient agar were screened for dome-shaped colony morphology on agar with sucrose and were found to be closely related by several criteria. The isolates were not pathogenic on apples or pears. They were characterized by a polyphasic approach including microbiological and API assays as well as fatty acid methyl ester analysis, DNA-DNA hybridization and DNA sequencing. For molecular classification, the 16S rRNA cistron and the conserved genes gpd and recA of these bacteria were investigated. Together with other taxonomic criteria, the results of these studies indicate that the bacteria belong to a novel separate species, which we propose to name Erwinia tasmaniensis sp. nov., with the type strain Et1/99(T) (=DSM 17950(T)=NCPPB 4357(T)). From DNA-DNA hybridization kinetics, microbiological characteristics and nucleotide sequence analyses, this species is related to pathogenic Erwinia species, but also to the epiphytic species Erwinia billingiae.

Molecular mechanisms underlying inducible cobalt and nickel resistance of a bacterial strain isolated from a Cuban serpentine deposit were investigated. This strain C-1 was assigned to Serratia marcescens by 16S rDNA analysis and DNA/DNA hybridization. Genes involved in metal resistance were identified by transposon mutagenesis followed by selection for cobalt- and nickel-sensitive derivatives. The transposon insertion causing the highest decrease in metal resistance was located in the ncrABC determinant. The predicted NcrA product was a NreB ortholog of the major facilitator protein superfamily and central for cobalt/nickel resistance in S. marcescens strain C-1. NcrA also mediated metal resistance in Escherichia coli and caused decreased accumulation of Co(II) and Ni(II) in this heterologous host. NcrB may be a regulatory protein. NcrC was a protein of the nickel–cobalt transport (NiCoT) protein family and necessary for full metal resistance in E. coli, but only when NcrA was also present. Without NcrA, NcrC caused a slight decrease in metal resistance and mediated increased accumulation of Ni(II) and Co(II). Because the cytoplasmic metal concentration can be assumed to be the result of a flow equilibrium of uptake and efflux processes, this interplay between metal uptake system NcrC and metal efflux system NcrA may contribute to nickel and cobalt resistance in this bacterium.

ABSTRACT The thermosensitive phenotype of strain CH31, a derivative of Corynebacterium ammoniagenes ATCC 6872, was allocated by cloning, sequencing, and genetic complementation to a single C→T exchange in the nrdE ( n ucleotide r e d uction) gene at nucleotide 1301. Protein modeling indicates the impaired surface hydrophobicity of NrdE(Ts) due to the S434F transition.

Bacteria were either isolated from leaf surfaces of Hedera helix or obtained from a culture collection in order to analyse their effect on barrier properties of isolated Hedera and Prunus laurocerasus cuticles. On the basis of the 16S rDNA sequences the genera of the six bacterial isolates from Hedera were identified as Pseudomonas sp., Stenotrophomonas sp. and Achromobacter. Water permeability of cuticles isolated from H. helix was measured before and after inoculation with the six bacterial strains. In addition water permeability of cuticles isolated from P. laurocerasus was measured before and after inoculation with the three bacterial strains Pseudomonas aeruginosa, Xanthomonas campestris and Corynebacterium fascians. Rates of water diffusing across isolated cuticles of both species significantly increased by up to 50% after inoculation with all bacterial strains. Obtained results show that epiphytic bacteria have the ability of increasing water permeability of Hedera and Prunus cuticles, which in turn should increase the availability of water and dissolved compounds in the phyllopshere. Consequently, living conditions in the habitat phyllosphere are improved. It can be concluded that the ability to change leaf surface properties will improve epiphytic fitness of leaf surface bacteria.

Corynebacterium ammoniagenes strain CH31 is thermosensitive due to a mutation in nucleotide reduction (nrdts). The strain was examined for nucleotide overproduction upon shifting the culture temperature to a range of elevated temperatures. No overproduction of NAD+ was detected in the control maintained at 27°C whereas NAD+ was accumulated extracellularily by strain CH31 at 37°C and at 40°C. As a result of the temperature shift, division-inhibited cells displayed only limited elongation. This is a characteristic morphological feature of cell-cycle-arrested coryneform bacteria. Ribonucleotide reductase (RNR) activity was inactivated immediately after the temperature shift in the NAD+-proficient cultures, leading presumably to an exhaustion of deoxyribonucleotide pools and impairment of DNA replication. In contrast to the low extracellular accumulation of NAD+, at the non-permissive temperature of 35°C a distinct capacity for intracellular nucleotide overproduction was revealed by a new method using nucleotide-permeable cells. The approach of shifting the culture temperature was applied successfully to the overproduction of taste-enhancing nucleotides in the presence of 10 μM Mn2+. Concomitant with a dramatic loss of viability, the thermosensitive mutant CH31 accumulated 5.3 g 5′-inosine monophosphate per liter following the addition of hypoxanthine as precursor for the salvage pathway.

The bacterial strain FLB300 was enriched with 3-fluorobenzoate as sole carbon source. Besides benzoate all isomeric monofluorobenzoates were utilized. Regioselective 1,2-dioxygenation rather than 1,6-dioxygenation yielded 4-fluorocatechol and minimized the production of toxic 3-fluorocatechol. Degradation of 4-fluorocatechol was mediated by reactions of ortho cleavage pathway activities. Chemotaxonomic and r-RNA data excluded strain FLB300 from a phylogenetically defined genus Pseudomonas and suggested its allocation to the alpha-2 subclass of Proteobacteria in a new genus of the Agrobacterium-Rhizobium branch.

Twenty-two indigenous yeasts isolated from leaves and fruits of German apple orchards were tentatively allocated by a polyphasic approach to ascomycetous and basidomycetous genera. In the laboratory screening, the isolates displayed only weak inhibitory activity when tested for their antagonistic activity against Venturia inaequalis (Cooke) Winter amend. Aderhold, the causal agent of apple scab. However, in greenhouse trials, three strains suppressed scab severity on seedlings of Malus domestica (Borkh.) cv. 'Golden Delicious' as efficient as fungicide controls. Colonization of the surface of apple leaves by the isolate H25 and its antagonistic activity towards conidia of the apple scab pathogen was demonstrated by scanning electron microscopy. Application of a concentration of 1.5 x 10(7) yeasts per ml on 9-year-old apple trees cv. 'Golden Delicious' in an experimental orchard in Dossenheim, Germany, once in May 1998 led to significant scab reduction by strain H 10 whereas the strains H 15, and H25 were less effective. The low performance of the antagonists observed in the season 1999 is not due to inefficient propagation since their establishment under field conditions and even spreading to other trees in the immediate neighbourhood of application was demonstrated using a culture method.

Cell division of the wild type strain Corynebacterium (formerly Brevibacterium) ammoniagenes ATCC 6872 which requires 1 μM Mn2+ for balanced growth was inhibited by addition of 20 mM hydroxyurea (HU) or 10 mM p-methoxyphenol (MP) to a Mn2+-supplemented fermentation medium at an appropriate time. Scanning electron microscopy (SEM) showed a restricted elongation characteristic of arrest of the cell cycle in coryneform bacteria. The cultures treated with HU or MP had, respectively, a fourfold or sixfold enhanced accumulation of NAD+ by a salvage biosynthetic pathway. An assay of nucleotide-permeable cells for ribonucleotide reductase activity using [3H-CDP] as substrate revealed a pre-early and complete decline of DNA precursor biosynthesis not found in the untreated control. Overproduction of NAD+ is an alternative to the conventional fermentation process using Mn2+ deficiency. A simple model is presented to discuss the metabolic regulation of the new process based on the presence of a manganese ribonucleotide reductase (Mn-RNR) in the producing strain.