Physiological responses of Arthrobacter sp. JQ-1 cell interfaces to co-existed di-(2-ethylhexyl) phthalate (DEHP) and copper.

Arthrobacter sp. JQ-1 can completely degrade 500 mg/L of DEHP within 3 days. The minimum inhibitory concentrations (MICs) of Cu2+ could reach 1.56 mM, however, 5.0 mg/L Cu2+ apparently inhibited DEHP degradation and bacterial growth. Consequently, JQ-1 was exposed to the DEHP-copper environment to verify the toxicity mechanism based on the physiological responses of cellular multiple interfaces (cellular surface, membrane and intracellular characteristics). The results showed the combination of 500 mg/L DEHP and 5.0 mg/L Cu2+ significantly decreased cell surface hydrophobicity (CSH) and the absolute value of zeta potential, which implied the bioavailability of DEHP was decreased. The cellular surface changes were mainly due to the interaction between Cu2+ and some functional groups (CH2, CH3, aromatic rings, and amide). The weakened proton-motive force (PMF) across the plasma membrane may interfere the formation and utilization of energy, which is not conducive to the repair process of cellular damages. In this study, Non-invasive micro-test technology (NMT) was applied to the research of combined toxicity of DEHP and heavy metal ions for the first time. DEHP-copper intensified K+ efflux and Ca2+ influx across the plasma membrane, which disturbed ion homeostasis of K+ and Ca2+ and might induce apoptosis and further inhibit DEHP degradation. The decline of intracellular esterase activity indicated that the metabolic capacity is apparently restrained. This study enhances our understanding of cellular different interface processes responding to combined pollutants.

The effect of uranium on bacterial viability and cell surface morphology using atomic force microscopy in the presence of bicarbonate ions.

Past disposal practices at nuclear production facilities have led to the release of liquid waste into the environment creating multiple radionuclide plumes. Microorganisms are known for the ability to interact with radionuclides and impact their mobility in soils and sediments. Gram-positive Arthrobacter sp. are one of the most common bacterial groups in soils and are found in large numbers in subsurface environments contaminated with radionuclides. This study experimentally analyzed changes on the bacteria surface at the nanoscale level after uranium exposure and evaluated the effect of aqueous bicarbonate ions on U(VI) toxicity of a low uranium-tolerant Arthrobacter oxydans strain G968 by investigating changes in adhesion forces and cell dimensions via atomic force microscopy (AFM). Experiments were extended to assess cell viability by the Live/Dead BacLight Bacterial Viability Kit (Molecular Probes) and quantitatively illustrate the effect of uranium exposure in the presence of varying concentrations of bicarbonate ions. AFM and viability studies showed that samples containing bicarbonate were able to withstand uranium toxicity and remained viable. Samples containing no bicarbonate exhibited deformed surfaces and a low height profile, which, in conjunction with viability studies, indicated that the cells were not viable.

Hydroxypropyl-ß-cyclodextrin-mediated alterations in cell permeability, lipid and protein profiles of steroid-transforming Arthrobacter simplex.

Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) enhances steroid 1-dehydrogenation biotransformation by Arthrobacter simplex. In this work, HP-ß-CD-induced improvement of A. simplex CPCC 140451 cell envelope permeability which had positive effects on the steroid bioconversion was confirmed by a comparative investigation which showed a lower dehydrogenase activity and higher cell permeability of the cells after being incubated with HP-ß-CD. Atomic force microscopy and transmission electron microscopy micrographs showed that HP-ß-CD altered the size, sharpness, and surface structure of the cell envelope. The analysis of lipid composition revealed that the proportion of extractable lipids decreased and the fatty acids profile was considerably altered. The contents of unsaturated fatty acids and long-chain fatty acids were reduced by 11.77 and 14.98%, respectively. The total leakage of protein level increased to 8%. Proteins belonging to the ATP-binding cassette superfamily and major facilitator superfamily were observed outside the cell. These alterations can explain the change of permeability on the molecular level under HP-ß-CD treatment. Results showed the material basis and mechanisms underlying the cellular changes, thus most likely contributing to the conversion rate in addition to cyclodextrins known effects on substrate solubility.

Influence of internal biofilm growth on residual permeability loss in aerobic granular membrane bioreactors.

Membrane fouling results in flux decline or transmembrane pressure drop increase during membrane bioreactor (MBR) operation. Physical and chemical cleanings are essential to keep an MBR operating at an appropriate membrane flux. Considerable residual membrane permeability loss that cannot be removed by conventional cleaning requires membrane replacement. This study demonstrates that an internal biofilm can develop inside a hollow-fiber membrane and can probably account for up to 58.9 and 81.3% of total membrane resistance for aerobic granular MBR operated in sequencing batch reactor (SBR) mode or continuous-fed mode, respectively. The Arthrobacter sp. (accession no. AM900505 in GenBank) corresponded to internal biofilm development by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis and the agar-plating technique. This study also identifies a single strain, Arthrobacter sp., generates the internal biofilm. The Arthrobacter sp. is a rod-shaped bacterium with a size close to that of membrane pores, and can secrete excess bound proteins, hence can penetrate and attach itself inside the membrane and grow. Internal biofilm growth could contribute significantly to membrane resistance during long-term MBR operation.

Production of acrylic acid from Acrylonitrile by immobilization of Arthrobacter nitroguajacolicus ZJUTB06-99.

Immobilized cells of Arthrobacter nitroguajacolicus ZJUTB06-99 capable of producing nitrilase was used for biotransformation of acrylonitrile to acrylic acid. Six different entrapment matrixes were chosen to search for a suitable support in terms of nitrilase activity. Ca-alginate proved to be more advantageous over other counterparts in improvement of the biocatalyst activity and bead mechanical strength. The effects of sodium alginate concentration, CaCl2 concentration, bead diameter, and ratio by weight of cells to alginate, on biosynthesis of acrylic acid by immobilized cells were investigated. Maximum activity was obtained under the conditions of 1.5% sodium alginate concentration, 3.0% CaCl2 concentration, and 2-mm bead size. The beads coated with 0.10% polyethylenimine (PEI) and 0.75% glutaraldehyde (GA) could tolerate more phosphate and decrease leakage amounts of cells from the gel. The beads treated with PEI/ GA could be reused up to 20 batches without obvious decrease in activities, which increased about 100% compared with the untreated beads with a longevity of 11 batches.

Azolla-Anabaena's behaviour in urban wastewater and artificial media--influence of combined nitrogen.

The results of using the nitrogen fixing symbiotic system Azolla-Anabaena to improve the quality of treated urban wastewater, particularly on what concerns phosphorus removal efficiencies (40-65%), obtained in continuous assays performed during the past few years and presented earlier, were very promising. Nevertheless, the presence of combined nitrogen in some wastewaters can compromise the treatment efficiency. The main goal of this work was to compare plants behaviour in wastewater and in mineral media with and without added nitrogen. Azolla filiculoides's specific growth rates in wastewater and in mineral media without added nitrogen or with low nitrate concentration were very similar (0.122 d(-1)-0.126 d(-1)), but decreased in the presence of ammonium (0.100 d(-1)). The orthophosphate removal rate coefficients were similar in all the growth media (0.210 d(-1)-0.232 d(-1)), but ammonium removal rate coefficient in wastewater was higher (0.117 d(-1)) than in mineral medium using that source of nitrogen (0.077 d(-1)). The ammonium present in wastewater, despite its high concentration (34 mg NL(-1)), didn't seem to inhibit growth and nitrogen fixation, however, in mineral media, ammonium (40 mg NL(-1)) was found to induce, respectively, 18% and 46% of inhibition.

Uptake of L-nicotine and of 6-hydroxy-L-nicotine by Arthrobacter nicotinovorans and by Escherichia coli is mediated by facilitated diffusion and not by passive diffusion or active transport.

The mechanism by which l-nicotine is taken up by bacteria that are able to grow on it is unknown. Nicotine degradation by Arthrobacter nicotinovorans, a Gram-positive soil bacterium, is linked to the presence of the catabolic megaplasmid pAO1. l-[(14)C]Nicotine uptake assays with A. nicotinovorans showed transport of nicotine across the cell membrane to be energy-independent and saturable with a K(m) of 6.2+/-0.1 microM and a V(max) of 0.70+/-0.08 micromol min(-1) (mg protein)(-1). This is in accord with a mechanism of facilitated diffusion, driven by the nicotine concentration gradient. Nicotine uptake was coupled to its intracellular degradation, and an A. nicotinovorans strain unable to degrade nicotine (pAO1(-)) showed no nicotine import. However, when the nicotine dehydrogenase genes were expressed in this strain, import of l-[(14)C]nicotine took place. A. nicotinovorans pAO1(-) and Escherichia coli were also unable to import 6-hydroxy-l-nicotine, but expression of the 6-hydroxy-l-nicotine oxidase gene allowed both bacteria to take up this compound. l-Nicotine uptake was inhibited by d-nicotine, 6-hydroxy-l-nicotine and 2-amino-l-nicotine, which may indicate transport of these nicotine derivatives by a common permease. Attempts to correlate nicotine uptake with pAO1 genes possessing similarity to amino acid transporters failed. In contrast to the situation at the blood-brain barrier, nicotine transport across the cell membrane by these bacteria was not by passive diffusion or active transport but by facilitated diffusion.

Microbiological degradation of pentane by immobilized cells of Arthrobacter sp.

The increasing production of several plastics such as expanded polystyrene, widely used as packaging and building materials, has caused the release of considerable amounts of pentane employed as an expanding agent. Today many microorganisms are used to degrade hydrocarbons in order to minimize contamination caused by several industrial activities. The aim of our work was to identify a suitable microorganism to degrade pentane. We focused our attention on a strain of Arthrobacter sp. which in a shake-flask culture produced 95% degradation of a 10% mixture of pentane in a minimal medium after 42 days of incubation at 20 degrees C. Arthrobacter sp. cells were immobilized on a macroporous polystyrene particle matrix that provides a promising novel support for cell immobilization. The method involved culturing cells with the expanded polystyrene in shake-flasks, followed by in situ growth within the column. Scanning electron microscopy analysis showed extensive growth of Arthrobacter sp. on the polymeric surface. The immobilized microorganism was able to actively degrade a 10% mixture of pentane, allowing us to obtain a bioconversion yield of 90% after 36 h. Moreover, in repeated-batch operations, immobilized Arthrobacter sp. cells were able to maintain 85-95% pentane degradation during a 2 month period. Our results suggest that this type of bioreactor could be used in pentane environmental decontamination.

[Ultrastructure of resting cells of some non-spore-forming bacteria]. / Tonkoe stroenie pokoiashchikhsia kletok nekotorykh nesporoobrazuiushchikh bakterii.

Using electron microscopy (ultrathin sections and freeze-fractures), we investigated the ultrastructure of the resting cells formed in the cultures of Micrococcus luteus, Arthrobacter globiformis, and Pseudomonas aurantiaca under conditions of prolonged incubation (up to 9 months). These resting cells included cyst-like forms that were characterized by complex cell structure and the following ultrastructural properties: (i) a thickened or multiprofiled cell wall (CW), typically made up of a layer of the preexisting CW and one to three de novo synthesized murein layers; (ii) a thick, structurally differentiated capsule; (iii) presence of large intramembrane particles (d = 180-270 A), occurring both on the PF and EF sides of the membrane fractures of M. luteus and A. globiformis; (iv) a peculiar structure of the cytoplasm, which was either fine-grained or lumpy (coarse-grained) in different parts of the cell population; and (v) a condensed nucleoid. Intense formation of cyst-like cells occurred in aged (2- to 9-month-old) bacterial cultures grown on diluted complex media or on nitrogen-, carbon-, and phosphorus-limited synthetic media, as well as in suspensions of cells incubated in media with sodium silicate. The general morphological properties, ultrastructural organization, and physiological features of cyst-like cells formed during the developmental cycle suggest that constitutive dormancy is characteristic of non-spore-forming bacteria.

The structure of resting bacterial populations in soil and subsoil permafrost.

The structure of individual cells in microbial populations in situ of the Arctic and Antarctic permafrost was studied by scanning and transmission electron microscopy methods and compared with that of cyst-like resting forms generated under special conditions by the non-spore-forming bacteria Arthrobacter and Micrococcus isolated from the permafrost. Electron microscopy examination of microorganisms in situ revealed several types of bacterial cells having no signs of damage, including "dwarf" curved forms similar to nanoforms. Intact bacterial cells in situ and frozen cultures of the permafrost isolates differed from vegetative cells by thickened cell walls, the altered structure of cytoplasm, and the compact nucleoid, and were similar in these features to cyst-like resting forms of non-spore-forming "permafrost" bacterial strains of Arthrobacter and Micrococcus spp. Cyst-like cells, being resistant to adverse external factors, are regarded as being responsible for survival of the non-spore-formers under prolonged exposure to subzero temperatures and can be a target to search for living microorganisms in natural environments both on the Earth and on extraterrestrial bodies.

[Microbiota of the Orchid rhizoplane]. / Mikrobiota poverkhnosti kornei Orkhidnykh.

Six bacterial strains isolated from the underground roots of the terrestrial orchid Calanthe vestita var. rubrooculata were found to belong to the genera Arthrobacter, Bacillus, Mycobacterium, and Pseudomonas. Strains isolated from the aerial roots of the epiphytic orchid Dendrobium moschatum were classified into the genera Bacillus, Curtobacterium, Flavobacterium, Nocardia, Pseudomonas, Rhodococcus, and Xanthomonas. The rhizoplane of the terrestrial orchid was also populated by cyanobacteria of the genera Nostoc and Oscillatoria, whereas that of the epiphytic orchid was populated by one genus, Nostoc. In orchids occupying different econiches the spectra of the bacterial genera revealed differed. The microbial complex of the terrestrial orchid rhizoplane differed from that of the surrounding soil.

[Synthesis of anabiosis autoinducers in non-spore-forming bacteria as a mechanism regulating their activity in soil and subsoil sedimentary rocks]. / Sintez autoinduktorov anabioza u nesporoobrazuiushchikh bakterii kak mekhanizm reguliatsii ikh aktivnosti v pochve i podpochvennykh osadochnykh porodakh.

Non-spore-forming bacteria of the genera Arthrobacter and Micrococcus, isolated from permafrost subsoil, were found to produce greater amounts of the d1 extracellular factor than closely related collection strains isolated from soil. The effect of this factor, responsible for cell transition to anabiosis, was not species-specific. Thus, the d1 crude preparation isolated from the culture liquid of the permafrost isolate Arthrobacter globiformis 245 produced an effect on the collection strain Arthrobacter globiformis B-1112 and also on Micrococcus luteus and Bacillus cereus. The crude d1 preparation from the permafrost isolate of Arthrobacter differed from the chemical analogue of this factor, 4n-hexylresorcinol, in the level of the induced cell response, which may have resulted from different cell sensitivity to various homologs of alkylhydroxybenzenes contained in the d1 preparation. Thus, additional evidence was obtained indicating that autoregulation of bacterial growth and development is implemented at the level of intercellular interactions in microbial communities. Abundant production of the d1 anabiosis-inducing factors by bacteria isolated from permafrost subsoil is probably a result of special antistress mechanisms responsible for the survival of these bacteria under extreme conditions of natural deep cooling.

Biochemical and phylogenetic analyses of psychrophilic isolates belonging to the Arthrobacter subgroup and description of Arthrobacter psychrolactophilus, sp. nov.

During our work on psychrophilic microorganisms we obtained a large collection of new isolates. In order to identify six of these, we examined their growth properties, cell wall compositions, and their 16S rRNA gene sequences. The results showed that all of the isolates are gram-positive, aerobic, contain lysine in their cell walls, and belong to the high mol% G+C Arthrobacter subgroup. Phylogenetic analysis of the 16S rRNA genes grouped five isolates obtained from a small geographical region into a monophyletic clade. Isolate B7 had a 16S rRNA sequence that was 94.3% similar to that of Arthrobacter polychromogenes and 94.4% similar to that of Arthrobacter oxydans. Primary characteristics that distinguish isolate B7 from the Arthrobacter type strain (Arthrobacter globiformis) and A. polychromogenes include lack of growth at 37 degrees C, growth at 0-5 degrees C, the ability to use lactose as a sole carbon source, and the absence of blue pigments. Because of these differences, isolate B7 was chosen as a type strain representing a new Arthrobacter species, Arthrobacter psychrolactophilus. The sixth isolate, LV7, differed from the other five because it did not have the rod/ coccus morphological cycle and was most closely related to Arthrobacter agilis.

Description of Arthrobacter creatinolyticus sp. nov., isolated from human urine.

Three strains of creatinine-hydrolysing bacteria isolated from human urine were characterized taxonomically. They were aerobic, non-spore-forming, Gram-positive rods with the peptidoglycan of the cell wall containing lysine. MK-8 and MK-9 were found to be the major types of menaquinone. The G + C content of the DNA was 66-67 mol%. The 16S rRNA sequence of one strain (GIFU 12498) was determined and aligned with other high-G + C-content Gram-positive rods from different genera. Following phylogenetic analysis, this strain was placed in the genus Arthrobacter. Arthrobacter protophormiae was the most closely related species in the phylogenetic tree, and this species also showed the highest sequence homology value (97%) with GIFU 12498. However, DNA-DNA hybridization indicated that GIFU 12498 did not belong to A. protophormiae (33.8 +/- 3.5% chromosomal similarity). The three urine strains belonged to one species because they shared more than 95% DNA-DNA similarity. It is proposed that these strains are placed in the genus Arthrobacter as a new species, Arthrobacter creatinolyticus sp. nov. The type strain of A. creatinolyticus is GIFU 12498, which has been deposited in the Japan Collection of Microorganisms (JCM) with the accession number JCM 10102.

A strain of Arthrobacter that tolerates high concentrations of nitrate.

A gram-positive strain identified as Arthrobacter globiformis CECT 4500, tolerant to up to 1 M nitrate, was isolated from the grounds of a munitions factory. Under strict aerobic conditions, this bacterium used a wide variety of C-sources to obtain the energy required for growth, which took place when the nitrate concentration in the medium was below 150 mM. Cells of this bacterium growing in the absence of nitrate were seen as individual cells or forming pairs, whereas cells grown in the presence of nitrate formed short filaments. With ethylene glycol as the C-source, optimal conditions for the full nitrate removal by Arthrobacter were established under laboratory conditions with wastewaters from the synthesis of dinitroethylene glycol.

Isolation and characterization of a fluorene-degrading bacterium: identification of ring oxidation and ring fission products.

An Arthrobacter sp. strain, F101, able to use fluorene as the sole source of carbon and energy, was isolated from sludge from an oil refinery wastewater treatment plant. During growth in the presence of fluorene, four major metabolites were detected and isolated by thin-layer chromatography and high-performance liquid chromatography. 9-Fluorenol, 9H-fluoren-9-one, and 3,4-dihydrocoumarin were identified by UV spectra, mass spectrometry, and 300-MHz proton nuclear magnetic resonance. The fourth metabolite has been characterized, but precise identification was not possible. Since strain F101 is not able to grow with fluorenone, two different pathways of fluorene biodegradation are suggested: one supports cell growth and produces 3,4-dihydrocoumarin as an intermediate and probably the unidentified metabolite, and the other produces 9-fluorenol and 9H-fluoren-9-one and appears to be a dead-end route.

Reclassification of two strains of Arthrobacter oxydans and proposal of Arthrobacter nicotinovorans sp. nov.

Arthrobacter oxydans DSM 419 and DSM 420 have chemical and microbiological properties that are consistent with assignment to the genus Arthrobacter. Both organisms have the lysine-alanine-threonine-alanine peptidoglycan type. DNA-DNA pairing studies indicated that A. oxydans DSM 419 should be reclassified as Arthrobacter ureafaciens and that A. oxydans DSM 420T forms the nucleus of a distinct genomic species. We propose that A. oxydans DSM 420 should be reclassified as Arthrobacter nicotinovorans sp. nov. The type strain is strain DSM 420.

Localization of the enantiozymes of 6-hydroxy-nicotine oxidase in Arthrobacter oxidans by electron immunochemistry.

During the course of growth of Arthrobacter oxidans, induction of the enantiozymes 6-hydroxy-D-nicotine oxidase and 6-hydroxy-L-nicotine oxidase occurred in the presence of DL-nicotine. Cryoultramicrotomed sections obtained from cells grown to stationary phase were gold immunolabeled. The results obtained demonstrate that both enzymes are localized in the cytoplasm.