Aerobic Methylobacteria Are Capable of Synthesizing Auxins

Obligately and facultatively methylotrophic bacteria with different pathways of C1 metabolism were found to be able to produce auxins, particularly indole-3-acetic acid (IAA), in amounts of 3–100 g/ml. Indole-3-pyruvic acid and indole-3-acetamide were detected only in methylobacteria with the serine pathway of C1 metabolism (Methylobacterium mesophilicumand Aminobacter aminovorans).The production of auxins by methylobacteria was stimulated by the addition of L-tryptophan to the growth medium and was inhibited by ammonium ions. The methylobacteria under study lacked tryptophan decarboxylase and tryptophan side-chain oxidase. At the same time, they were found to contain several aminotransferases. IAA is presumably synthesized by methylobacteria through indole-3-pyruvic acid.     

Physiological and Biochemical Analysis of the Transformants of Aerobic Methylobacteria Expressing the dcmA Gene of Dichloromethane Dehalogenase

Transformants of Methylobacterium dichloromethanicum DM4 (DM4-2cr^–/pME 8220 and DM4-2cr^–/pME8221) and of Methylobacterium extorquens AM1 (AM1/pME8220 and AM1/pME8221) that express the dcm A gene of dichloromethane dehalogenase undergo lysis when incubated in the presence of dichloromethane and are sensitive to acidic shock. The lysis of the transformants was found to be related neither to the accumulation of Cl^– ions, CH₂O, or HCOOH, nor to the impairment of glutathione synthesis or to the disturbance of intracellular pH homeostasis. The (exo^–) Klenow fragment–mediated incorporation of [-³²P]dATP into the DNA of the transformants DM4-2cr^–/pME8220 and AM1/pME8220 was considerably greater when the transformed cells were incubated with CH₂Cl₂ than when they were incubated with CH₃OH, indicating the occurrence of a significant increase in the total length of gaps. At the same time, the strain AM1 (which lacks dichloromethane dehalogenase) and the dichloromethane-degrading strain DM4 incubated with CH₂Cl₂ showed an insignificant increase in the total length of the gaps. The transformed cells are likely to lyse due to the relatively inefficient repair of DNA lesions that are induced in response to the alkylating action of S-chloromethylglutathione, an intermediate product of CH₂Cl₂ degradation. The data obtained suggest that the bacterial mineralization of dichloromethane requires an efficient DNA repair system.     

The Biology of Methylobacteria Capable of Degrading Halomethanes

Recent data on the biology of aerobic methylotrophic bacteria capable of utilizing toxic halogenated methane derivatives as sources of carbon and energy are reviewed, with particular emphasis on the taxonomic, physiological, and biochemical diversity of mono- and dihalomethane-degrading methylobacteria and the enzymatic and genetic aspects of their primary metabolism. The initial steps of chloromethane dehalogenation to formate and HCl through a methylated corrinoid and methyltetrahydrofolate are catalyzed by inducible cobalamin methyl transferase, made up of two proteins (CmuA and CmuB) encoded by the cmuA and cmuB genes. At the same time, the primary dehalogenation of dichloromethane to formaldehyde and HCl is catalyzed by cytosolic glutathione transferase with S-chloromethylglutathione as an intermediate. The latter enzyme is encoded by the structural dcmA gene and is under the negative control of the regulatory dcmR gene. In spite of considerable progress in the study of halomethane dehalogenation, some aspects concerning the structural and functional organization of this process and its regulation remain unknown, including the mechanisms of halomethane transport, the release of toxic dehalogenation products (S-chloromethylglutathione, CH₂O, and HCl) from cells, and the maintenance of intracellular pH. Of particular interest is a quantitative evaluation of the ecophysiological role of aerobic methylobacteria in the mineralization of halomethanes and the protection of the biosphere from these toxic pollutants.     

Excretion of Sugars by Chlorella Species Capable and Incapable of Symbiosis with Hydra viridis

The excretion of some sugars (maltose, glucose, and glucose-6-phosphate) was studied at pH 2.5–6.0 in 38 strains of Chlorella belonging to 15 species of which 7 are capable and 8 incapable of symbiosis with Hydra viridis. A high rate of maltose excretion below pH 4.0 (Cernichiari et al., 1969) was found only in C. vulgaris (non-symbiotic) and C. mirabilis (non-symbiotic). The other Chlorella species are characterized by quite different patterns of sugar excretion. C. spec. (= “C. paramecii”; symbiotic) excretes very high amounts of maltose in the whole range from pH 2.5–6.0. C. kessleri (symbiotic), C. luteoviridis (symbiotic), and C. fusca var. fusca (non-symbiotic) show a predominant excretion of glucose-6-phosphate from pH 2.5–6.0. Some strains also exhibit a high excretion of glucose above pH 4.0 (C. spec. = “C. paramecii”) or below pH 3.0 (C. fusca var. vacuolata). Several species, e.g. C. saccharophila var. saccharophila (symbiotic), C. sorokiniana (non-symbiotic), and C. protothecoides (symbiotic), excrete only very small amounts of sugars. There is no obvious correlation between sugar excretion and the ability or inability of the Chlorella species to form stable symbioses with Hydra viridis.     

Identification of Soil Microbes Capable of Utilizing Cellobiosan

Approximately 100 million tons of anhydrosugars, such as levoglucosan and cellobiosan, are produced through biomass burning every year. These sugars are also produced through fast pyrolysis, the controlled thermal depolymerization of biomass. While the microbial pathways associated with levoglucosan utilization have been characterized, there is little known about cellobiosan utilization. Here we describe the isolation and characterization of six cellobiosan-utilizing microbes from soil samples. Each of these organisms is capable of using both cellobiosan and levoglucosan as sole carbon source, though both minimal and rich media cellobiosan supported significantly higher biomass production than levoglucosan. Ribosomal sequencing was used to identify the closest reported match for these organisms: Sphingobacterium multivorum, Acinetobacter oleivorans JC3-1, Enterobacter sp SJZ-6, and Microbacterium sps FXJ8.207 and 203 and a fungal species Cryptococcus sp. The commercially-acquired Enterobacter cloacae DSM 16657 showed growth on levoglucosan and cellobiosan, supporting our isolate identification. Analysis of an existing database of 16S rRNA amplicons from Iowa soil samples confirmed the representation of our five bacterial isolates and four previously-reported levoglucosan-utilizing bacterial isolates in other soil samples and provided insight into their population distributions. Phylogenetic analysis of the 16S rRNA and 18S rRNA of strains previously reported to utilize levoglucosan and our newfound isolates showed that the organisms isolated in this study are distinct from previously described anhydrosugar-utilizing microbial species.     

Method for Performing Aerobic Plate Counts of Anhydrous Cosmetics Utilizing Tween 60 and Arlacel 80 as Dispersing Agents

An aqueous diluent containing Tween 60 and Arlacel 80 gave greater recovery of microorganisms when compared with two common diluents as determined by aerobic plate count of inoculated anhydrous cosmetics. The greater recovery was caused by better dispersion of the anhydrous cosmetics in the diluents.     

Effects of Bacterial Host and Dichloromethane Dehalogenase on the Competitiveness of Methylotrophic Bacteria Growing with Dichloromethane

Methylobacterium sp. strain DM4 and Methylophilus sp. strain DM11 can grow with dichloromethane (DCM) as the sole source of carbon and energy by virtue of homologous glutathione-dependent DCM dehalogenases with markedly different kinetic properties (the k_cat values of the enzymes of these strains are 0.6 and 3.3 s⁻¹, respectively, and the K_m values are 9 and 59 μM, respectively). These strains, as well as transconjugant bacteria expressing the DCM dehalogenase gene (dcmA) from DM11 or DM4 on a broad-host-range plasmid in the background of dcmA mutant DM4-2cr, were investigated by growing them under growth-limiting conditions and in the presence of an excess of DCM. The maximal growth rates and maximal levels of dehalogenase for chemostat-adapted bacteria were higher than the maximal growth rates and maximal levels of dehalogenase for batch-grown bacteria. The substrate saturation constant of strain DM4 was much lower than the K_m of its associated dehalogenase, suggesting that this strain is adapted to scavenge low concentrations of DCM. Strains and transconjugants expressing the DCM dehalogenase from strain DM11, on the other hand, had higher growth rates than bacteria expressing the homologous dehalogenase from strain DM4. Competition experiments performed with pairs of DCM-degrading strains revealed that a strain expressing the dehalogenase from DM4 had a selective advantage in continuous culture under substrate-limiting conditions, while strains expressing the DM11 dehalogenase were superior in batch culture when there was an excess of substrate. Only DCM-degrading bacteria with a dcmA gene similar to that from strain DM4, however, were obtained in batch enrichment cultures prepared with activated sludge from sewage treatment plants.     

Enhanced Resistance of Pea Plants to Oxidative Stress Caused by Paraquat during Colonization by Aerobic Methylobacteria

The influence of colonization of the pea (Pisum sativum L.) by aerobic methylobacteria of five different species (Methylophilus flavus Ship, Methylobacterium extorquens G10, Methylobacillus arboreus Iva, Methylopila musalis MUSA, Methylopila turkiensis Side1) on plant resistance to paraquat-induced stresses has been studied. The normal conditions of pea colonization by methylobacteria were characterized by a decrease in the activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidases) and in the concentrations of endogenous H₂O₂, proline, and malonic dialdehyde, which is a product of lipid peroxidation and indicator of damage to plant cell membranes, and an increase in the activity of the photosynthetic apparatus (the content of chlorophylls а, b and carotenoids). In the presence of paraquat, the colonized plants had higher activities of antioxidant enzymes, stable photosynthetic indices, and a less intensive accumulation of the products of lipid peroxidation as compared to noncolonized plants. Thus, colonization by methylobacteria considerably increased the adaptive protection of pea plants to the paraquat-induced oxidative stress.     

Cyclin-dependent Kinases Participate in Death of Neurons Evoked by DNA-damaging Agents

Previous reports have indicated that DNA-damaging treatments including certain anticancer therapeutics cause death of postmitotic nerve cells both in vitro and in vivo. Accordingly, it has become important to understand the signaling events that control this process. We recently hypothesized that certain cell cycle molecules may play an important role in neuronal death signaling evoked by DNA damage. Consequently, we examined whether cyclin-dependent kinase inhibitors (CKIs) and dominant-negative (DN) cyclin-dependent kinases (CDK) protect sympathetic and cortical neurons against DNA-damaging conditions. We show that Sindbis virus–induced expression of CKIs p16^ink4, p21^waf/cip1, and p27^kip1, as well as DN-Cdk4 and 6, but not DN-Cdk2 or 3, protect sympathetic neurons against UV irradiation– and AraC-induced death. We also demonstrate that the CKIs p16 and p27 as well as DN-Cdk4 and 6 but not DN-Cdk2 or 3 protect cortical neurons from the DNA damaging agent camptothecin. Finally, in consonance with our hypothesis and these results, cyclin D1–associated kinase activity is rapidly and highly elevated in cortical neurons upon camptothecin treatment. These results suggest that postmitotic neurons may utilize Cdk4 and 6, signals that normally control proliferation, to mediate death signaling resulting from DNA-damaging conditions.     

The Fractionation of Chlorine Isotopes by the Aerobic Methylotrophic Bacterium Methylobacterium dichloromethanicum Grown on Dichloromethane

Methylobacterium dichloromethanicum was found to be able to utilize dichloromethane (DCM) as the source of carbon and energy with the production of biomass, CO₂, and HCl. A comparative analysis of the abundances of the major DCM isotopomers ³⁵Cl₂ ¹²C¹H₂, ³⁵Cl³⁷Cl¹²C¹H₂, and ³⁷Cl₂ ¹²CH₂1H₂ made it possible to estimate the fractionation of chlorine isotopes during the bacterial metabolism of DCM. The kinetic chlorine isotope effects for ³⁵Cl³⁷Cl¹²C¹H₂ (m/z 86) and ³⁷Cl₂ ¹²C¹H₂ (m/z 88) relative to ³⁵Cl₂ ¹²C¹H₂ (m/z 84) were characterized by _86/84 = 1.006 ± 0.002 and _88/84 = 1.023 ± 0.003, respectively. The inference is made that the growth of M. dichloromethanicum on DCM is accompanied by the mass-independent fractionation of the DCM isotopomers.     

Isolation, Characterization and Phylogenetic Analysis of an Aerobic Bacterium Capable of Degrading Bensulfuronmethyl

Summary An aerobic bacterium named strain BH was isolated from soil samples based on its bensulfuronmethyl-degrading characteristics using continuous enrichment cultures. The cells of the strain were non-motile, gram-positive short rods. Colonies formed on agar medium were round, smooth, sticky, white-yellow in colour and of butyrous consistency. Analyses of nutritional utilization in Biolog microplates, conventional phenotypic characteristics and 16S rRNA gene sequencing were consistent with assigning strain BH to the genus Brevibacterium. Growth of the cells and their ability to degrade bensulfuronmethyl were simultaneously monitored under different liquid medium conditions during 7 days of incubation. They degraded bensulfuronmethyl from 100 to 70.6 mg l⁻¹ in mineral M9 medium and exhibited more effective degradation in the presence of yeast extract, completely removing an initial concentration of 100 mg l⁻¹ and at best 80% of an initial concentration of 200 mg l⁻¹. Further studies are required to determine the potential use of the isolate in the disposal of bensulfuronmethyl residues in agriculture and industry.     

Comparative Analysis of Mono- and Bifunctional Chorismate Synthases in <Emphasis Type="Italic">Escherichia coli</Emphasis> Cells Capable and Incapable of Phenylalanine Production

The activity of chorismate synthase, the terminal enzyme of the common aromatic pathway, is absolutely dependent on reduced flavin mononucleotide. The bifunctional chorismate synthase of Saccharomyces cerevisiae (product of the ARO2 gene) can reduce flavin in a reaction that involves NADPH, in contrast to the monofunctional chorismate synthase of Escherichia coli (product of the _aro C gene). The latter enzyme does not have the capacity for flavin reduction, and its activity therefore depends on the flavin reductase function of the cell. Chemical synthesis of the structural part of the ARO2 gene that involved the substitution of rare E. coli codons was performed for an in vivo comparison of the two types of chorismate synthase. ARO2 expression was tested in the T7 system, and isogenic E. coli strains TG1Δ _aro CP_tac-ARO2 and TG1Δ _aro CP_tac- _aro C were obtained. Comparative analysis of proteins from the cell extracts of these strains and in silico assessment of hybrid RBS efficiency showed that the level of AroC protein synthesis in TG1Δ _aro CP_tac- _aro C was higher than the level of ARO2 synthesis in the TG1Δ _aro CP_tac-ARO2 cells. The introduction of Ptac-ARO2 and Ptac- _aro C modifications led to complete recovery of the growth of the aromatic auxotroph TG1Δ _aro C on minimal mineral medium supplemented with glucose and restored phenylalanine production in the E. coli strain DV1017Δ _aro C, which lacked chorismate synthase activity. The similar positive effects of P_tac- _aro C and P_tac-ARO2 on phenylalanine biosynthesis in the DV1017ΔtyrR strain, in which chorismate synthase played a “bottleneck” role, indicated the absence of a limiting effect of reduced flavin on monofunctional chorismate synthase overexpressed in E. coli cells.     

Effects of Oxygen-Releasing Materials on Aerobic Bacterial Degradation Processes

The aerobic microbial degradation of p-nitrophenol (PNP) and phenol was examined under oxygen-limiting conditions in the presence of three known oxygen-releasing materials: (1) polyvinylidene chloride-encapsulated sodium percarbonate; (2) REGENESIS oxygen-releasing compound (magnesium peroxide); and (3) PermeOx® solid peroxygen (calcium peroxide). The degradation of PNP or phenol in buffered solutions was measured in 40-mL reaction chambers containing 25 to 300 mg of oxygen-releasing materials in the presence of immobilized chemical-degrading bacteria. Radiometric studies were used to determine total chemical mineralization and material balances of ¹⁴C-residues. Degradation of PNP and phenol increased in proportion to both the supplemented amount and the relative oxygen content of each oxygen-releasing material. Comparison of actual to theoretical degradation, based on stoichiometry of balanced equations for mineralization, showed that chemical uptake (primary degradation) at lower concentrations of oxygen-releasing materials was two-fold higher than could be mineralized theoretically. Radiometric studies confirmed the relationships between chemical uptake, cellular ¹⁴C-residues, mineralization, and the concentration of oxygen-releasing material. Further studies with PermeOx(identified supplementation levels required to support aerobic bacterial mineralization of PNP and phenol at levels similar to those of aerobic controls. These results show how the concentration of oxygen-releasing materials affects the rate and extent of aerobic chemical under oxygen-limiting conditions.     

非诱变剂对姐妹染色单体交换的诱导效应

检测了16种非诱变剂对大麦根端分生细胞姐妹染色单体交换(SCE)的影响及其中9种成份对人外周血淋巴细胞SCE的影响。在大麦细胞中,用高浓度的葡萄糖、氯化钠、维生素、氨基酸及脱氧核糖核苷三磷酸(dNTP)处理均能显著提高SCE频率。维生素、氨基酸和dNTP对人外周血淋巴细胞中的SCE频率也有显著影响。实验结果表明,不只是诱变剂能提高SCE频率,非诱变剂在一定高剂量时也能提高SCE频率。非诱变剂影响SCE具有特殊的S CE增长曲线,SCE频率达一定值后不再随处理浓度的增大而提高。