Cells of Pseudomonas putida and Enterobacter sp. adapt to toxic organic compounds by increasing their size

The phenol-degrading solvent-tolerant bacterium Pseudomonas putida P8 changed its cell shape when grown in the presence of aromatic compounds such as phenol and 4-chlorophenol. The sizes of cells that had been growing after addition of different concentrations of the toxic compounds were measured using a coulter counter that calculates the sizes of the rod-shaped bacteria to diameters of virtual spheres. The cells showed an increase in the diameter depending on the toxic effects of the applied concentrations of both solvents. The same effect was measured for an alkanol degrading bacterium, Enterobacter sp. VKGH12, in the presence of n-butanol. The reaction of the cells to different concentrations of n-butanol was examined by scanning electron microscopy. With this technique it could be shown that the size of the bacteria increased with increasing concentrations of n-butanol. These changes in cell size were dependent on the cellular activity and occurred only after addition of non-lethal concentrations. In the presence of lethal concentrations that completely inhibited cell growth, the cell sizes were similar to those of cells without intoxication. Taking into account the mathematical formula for spherical and cylindrical diameter and surface, respectively, the cells reacted to the presence of organic solvents by decreasing the ratio between surface and volume of the cells and therefore reducing their relative surfaces. As the cell surface and especially the cytoplasmic membrane are the major targets for the toxic effects of membrane-active compounds, this reduction of the relative surface represents an adaptive response to the presence of such compounds.     

Influence of phenol, p-cresol and indole on growth and survival of intestinal lactic acid bacteria

Some intestinal bacteria can produce many genotoxic, mutagenic and carcinogenic substances. The major products of the bacterial aromatic amino acids fermentation-phenolic and indolic compounds which are responsible for colon cancer development are accumulated in the colon. The effect of phenol, p-cresol and indole (2, 20 and 100 microg/ml doses) on growth and survival of four strains of intestinal lactic acid bacteria was studied. Growth of bacteria was not affected by any of the concentrations of phenol and p-cresol tested. The growth of 2 strains was slightly inhibited by 100 microg/ml of indole. There was no influence of phenol and p-cresol on survival of lactic bacteria until 120 h and specific reaction to carcinogens depending on strain was observed after that incubation time. Indole concentrations 20 and 100 microg/ml appeared to be toxic for all tested strains but just after 24, 48 or 72 h of incubation depending on the strain. In total, 2 microg/ml of indole had a very little effect.     

Growth and physiological response of Arthrobacter protophormiae RKJ100 toward higher concentrations of o-nitrobenzoate and p-hydroxybenzoate

Bioremediation of sites that are heavily contaminated with pollutant chemicals is a challenge as most of the microorganisms cannot tolerate higher concentrations of toxic compounds. Only a few strains of the genus Pseudomonas have been studied for their tolerance toward the higher concentrations of aromatic pollutant compounds, a phenomenon that is accompanied by various physiological changes. In the present study we have characterized the growth response and physiological changes (adaptations) of a Gram-positive bacterium, Arthrobacter protophormiae RKJ100, toward the higher concentrations of two aromatic compounds, viz. o-nitrobenzoate (ONB) and p-hydroxybenzoate (PHB). Arthrobacter protophormiae RKJ100 could utilize 30 mM ONB and 50 mM PHB as sole sources of carbon and energy. It was capable of growth on higher concentrations of ONB (up to 200 mM) and PHB (up to 150 mM) when the cells were pre-exposed to lower concentrations of these compounds. The adaptive responses shown by the organism during growth on higher concentrations of these compounds were evident from significant changes in cellular fatty acid profiles. In addition, Bacterial Adhesion To Hydrocarbon (BATH) assay and scanning electron microscopy showed substantial increase in cell surface hydrophobicity and decrease in cell size of A. protophormiae RKJ100 when grown on ONB and PHB as compared to succinate-grown cells.     

Initial Stages of 2,4,6-Trinitrotoluene Transformation by Microorganisms

Screening of a wide range of microorganisms (32 strains) isolated from various anthropogenic and natural environments and of a number of collection strains showed that the early stages of 2,4,6-trinitrotoluene (TNT) transformation by the majority of the strains studied resulted in the formation of hydroxylaminodinitrotoluenes (HADNTs). The levels of HADNTs were in a number of cases comparable to the initial TNT level. The alternative reductive attack on TNT through the reduction of the aromatic ring was not characteristic of most of the prokaryotes studied. The susceptibility to the toxic effect of TNT was different for gram-positive and gram-negative bacteria.     

Initial stages of 2,4,6-trinitrotoluene transformation by microorganisms

Screening of a wide range of microorganisms (32 strains) isolated from various anthropogenic and natural environments and of a number of collection strains showed that the early stages of 2,4,6-trinitrotoluene (TNT) transformation by the majority of the strains studied resulted in the formation of hydroxylaminodinitrotoluenes (HADNTs). The levels of HADNTs were in a number of cases comparable to the initial TNT level. The alternative reductive attack at TNT through the reduction of the aromatic ring was not characteristic of most of the prokaryotes studied. The susceptibility to the toxic effect of TNT was different for gram-positive and gram-negative bacteria.     

Comparative study of the effect of selected agrochemical products on Steinernema feltiae (Rhabditida: Steinernematidae)

The effect of three neurotoxic insecticides, three photosynthetic inhibitor herbicides and three enzymatic inhibitor herbicides on infective juveniles (IJs) of Steinernema feltiae Rioja (native) and ENTONEM® (commercial) strains were evaluated after a 48-h exposure at field tank concentrations and overnight treatment in mQ-water, using Spodoptera littoralis as target. Nematode survival was not affected by acetyl-cholinesterase inhibitors chlorpyrifos and pirimicarb, although chlorpyrifos seriously reduced their virulence. Both nematode strains showed differential sensitivity to cypermethrin, which affects the sodium channels of the nerve membrane, with the ENTONEM® strain being more tolerant than Rioja strain. However, these chemicals showed a strong sublethal effect on the nematode reproductive potential, limiting seriously their possible recycling in the field. Herbicides showed differential toxic effects on nematode survival. The commercial strain was tolerant to enzymatic inhibitor herbicides, whereas tribenuron and chlorsulfuron reduced Rioja strain survival. However, photosynthetic inhibitor herbicides severely affected survival of both nematode strains, with the Rioja strain being more sensitive. Sublethal effects on both nematode strains were observed only after exposition to terbutryn+chlortoluron+triasulfuron, increasing the time to kill insect larvae. These results are useful to optimize EPN dosages and to estimate their field recycling.     

Growth-inhibiting effects of concentrations of fusaric acid on the growth of Bacillus mojavensis and other biocontrol Bacillus species

AIMS: To determine the effects of concentrations of fusaric acid on the growth of several strains of the biocontrol bacterial endophyte Bacillus mojavensis and other species within the Bacillus subtilis group, as well as the genetic relationships within this small group of Gram-positive bacteria, and their antagonisms to Fusarium verticillioides, which produce fusaric acid. METHODS AND RESULTS: The growth of 50 Bacillus strains and species were tested at two concentrations of fusaric acid determined in maize infected by an isolate of F. verticillioides. Molecular characterizations of the strains and species of bacteria were determined with an automated ribotyper. The growth of bacteria measured under both concentrations with an automated turbidometer, Bioscreen, indicated that fusaric acid was toxic to most strains of the bacterial endophyte B. mojavensis. However, the effects of these two concentrations on other Bacillus species varied in that fusaric acid was either bacteriocidal or bacteriostatic to most species. CONCLUSIONS: These data indicate that the concentrations of fusaric acid are inhibitory to the growth of most Bacillus species, some of which are used as biocontrol agents. This suggests that the endophytic and saprophytic states of F. verticillioides and other Fusarium species cannot be controlled by fusaric-acid-sensitive Bacillus species. SIGNIFICANCE AND IMPACT OF STUDY: Mycotoxic Fusarium species, such as F. verticillioides, are competitive because all produce fusaric acid, which is inhibitory to biocontrol bacteria, and mutants tolerant to fusaric acid must be developed in order to be effective on biocontrol bacteria.     

Isolation and initial characterization of bacteria growing on tetralin

Eight strains of bacteria utilizing tetralin as sole source of carbon and energy have been obtained. Four strains have been selected from culture collections. The others were isolated from hydrocarbon-polluted areas. The newly isolated strains belong to the genera Acinetobacter, Arthrobacter and Moraxella. Most of the selected strains were able to grow on other aromatic hydrocarbons, but none of them grew on cyclohexane. Tetralin-utilizing organisms were difficult to isolate and cultivate, because tetralin was toxic to the cells at concentrations above 15 l/l. Consequently tetralin was supplied either via the vapour phase or an organic solvent/water two-phase system was employed.Abbreviations FC 40 fluorocompound 40 - DBP dibutylphthalate - DEP diethylphthalate - DOP dioctylphthalate     

Solvent tolerance in Gram-negative bacteria

Bacteria have been found in all niches explored on Earth, their ubiquity derives from their enormous metabolic diversity and their capacity to adapt to changes in the environment. Some bacterial strains are able to thrive in the presence of high concentrations of toxic organic chemicals, such as aromatic compounds, aliphatic alcohols and solvents. The extrusion of these toxic compounds from the cell to the external medium represents the most relevant aspect in the solvent tolerance of bacteria, however, solvent tolerance is a multifactorial process that involves a wide range of genetic and physiological changes to overcome solvent damage. These additional elements include reduced membrane permeabilization, implementation of a stress response programme, and in some cases degradation of the toxic compound. We discuss the recent advances in our understanding of the mechanisms involved in solvent tolerance.     

Distribution of aldoxime dehydratase in microorganisms

The distribution of phenylacetaldoxime-degrading and pyridine-3-aldoxime-degrading ability was examined with intact cells of 975 microorganisms, including 45 genera of bacteria, 11 genera of actinomyces, 22 genera of yeasts, and 37 genera of fungi, by monitoring the decrease of the aldoximes by high-pressure liquid chromatography. The abilities were found to be widely distributed in bacteria, actinomyces, fungi, and some yeasts: 98 and 107 strains degraded phenylacetaldoxime and pyridine-3-aldoxime, respectively. All of the active strains exhibited not only the aldoxime-dehydration activity to form nitrile but also nitrile-hydrolyzing activity. On the other hand, all of 19 nitrile-degrading microorganisms (13 species, 7 genera) were found to exhibit aldoxime dehydration activity. It is shown that aldoxime dehydratase and nitrile-hydrolyzing activities are widely distributed among 188 aldoxime and 19 nitrile degraders and that the enzymes were induced by aldoximes or nitriles.     

Distribution of Aldoxime Dehydratase in Microorganisms

The distribution of phenylacetaldoxime-degrading and pyridine-3-aldoxime-degrading ability was examined with intact cells of 975 microorganisms, including 45 genera of bacteria, 11 genera of actinomyces, 22 genera of yeasts, and 37 genera of fungi, by monitoring the decrease of the aldoximes by high-pressure liquid chromatography. The abilities were found to be widely distributed in bacteria, actinomyces, fungi, and some yeasts: 98 and 107 strains degraded phenylacetaldoxime and pyridine-3-aldoxime, respectively. All of the active strains exhibited not only the aldoxime-dehydration activity to form nitrile but also nitrile-hydrolyzing activity. On the other hand, all of 19 nitrile-degrading microorganisms (13 species, 7 genera) were found to exhibit aldoxime dehydration activity. It is shown that aldoxime dehydratase and nitrile-hydrolyzing activities are widely distributed among 188 aldoxime and 19 nitrile degraders and that the enzymes were induced by aldoximes or nitriles.     

The photobioluminometer,an instrument for the study of ecological factors affecting photosynthesis

An instrument for measurement of bioluminescence and photosynthesis is described. It may be used to detect chemicals toxic to luminous bacteria or to measure photosynthetic oxygen production of algae in mixed culture with bacteria. The latter technique has been used to study the effect on algal photosynthesis of environmental factors such as light quality, temperature, and salinity, and to study the factors affecting chlorophyll synthesis. The technique is ideal for rapid detection of photosynthesis-inhibiting herbicides and other toxic substances in water.     

Phenylurea herbicides induce cytogenetic effects in Chinese hamster cell lines

The intensive use of herbicides over the last few decades has caused a general increase of environmental pollution. It is thus very important to evaluate the possible genotoxic properties of these chemical compounds as well as identifying their mode of action. Phenylurea herbicides are selective agents widely used for the control of infestant plants. Of these herbicides, which are widely used in agriculture, we analysed four of the less intensively studied molecules. More precisely, we investigated the genotoxic effects of fenuron, chlorotoluron, diuron, and difenoxuron by analyses of chromosomal aberrations (CAs) and sister chromatid exchange (SCE) in exposed mammalian cells. We used the Chinese hamster ovary (CHO) and epithelial liver (CHEL) cell lines, endowed with the absence or the presence, respectively, of an enzymatic system to activate pro-mutagenic compounds. Our results show that all herbicides tested induce, at high concentrations, an increasing number of CAs in non-metabolising CHO cells. Instead, in the exposed CHEL cell line, the four herbicides induced CAs also at the lowest dose-level. In the CHEL cells, a statistically significant increase of SCE was also observed. The phenylurea herbicides showed direct genotoxic activity, but the cytogenetic effects were greatly enhanced after metabolic conversion. These data, together with other information on phenylurea herbicides, are of great interest from the environmental point of view, and for human health. In fact, intensive use of herbicides contaminates soil, surface water, groundwater and agricultural products, and thus should be taken in particular consideration not only for those initiatives to specifically protect exposed workers, but also to safeguard the health of consumers of agricultural products.     

Photocatabolism of Aromatic Compounds by the Phototrophic Purple Bacterium Rhodomicrobium vannielii

The phototrophic purple non-sulfur bacterium Rhodomicrobium vannielii grew phototrophically (illuminated anaerobic conditions) on a variety of aromatic compounds (in the presence of CO₂). Benzoate was universally photocatabolized by all five strains of R. vannielii examined, and benzyl alcohol was photocatabolized by four of the five strains. Catabolism of benzyl alcohol by phototrophic bacteria has not been previously reported. Other aromatic substrates supporting reasonably good growth of R. vannielii strains were the methoxylated benzoate derivatives vanillate (4-hydroxy-3-methoxybenzoate) and syringate (4-hydroxy-3,5-dimethoxybenzoate). However, catabolism of vanillate and syringate led to significant inhibition of bacteriochlorophyll synthesis in R. vannielii cells, eventually causing cultures to cease growing. No such effect on photopigment synthesis in cells grown on benzoate or benzyl alcohol was observed. Along with a handful of other species of anoxygenic phototrophic bacteria, the ability of the species R. vannielii to photocatabolize aromatic compounds indicates that this organism may also be ecologically significant as a consumer of aromatic derivatives in illuminated anaerobic habitats in nature.     

Effect of trace elements on surface hydrophobicity and adherence of Escherichia coli to uroepithelial cells

Trace elements have significant effect on the physiology of bacteria. Variation in the concentration of trace elements may affect the expression of virulence by microorganisms. The effect of trace elements on hydrophobicity and adherence of E.coli to uroepithelial cells was studied. Increasing concentrations of Ca2+, Mg2+, Fe3+ and Zn2+ significantly decreased the surface hydrophobicity. Toxic trace elements like Co2+, Cu2+, Mn2+ and Ni2+ did not alter surface hydrophobicity. With regards to adherence of E.coli to uroepithelial cells, only Mg2+ had significant effect. Toxic trace elements decreased the rate of cell adherence. The pathogenic strains of E.coli showed higher surface hydrophobicity and better cell adherence compared to the nonpathogenic strains. There was good correlation between surface hydrophobicity and cell adherence at higher concentrations (0.1 to 0.2mM) of Fe2+ and Zn2+. The results indicated that trace elements can significantly affect surface hydrophobicity and adherence of E.coli to uroepithelial cells. Such effect may have a significant impact on the initial stages of bacterial infection.     

Nitrile hydrolysing activities of deep-sea and terrestrial mycolate actinomycetes

Nitrile metabolising actinomycetes previously recovered from deep-sea sediments and terrestrial soils were investigated for their nitrile transforming properties. Metabolic profiling and activity assays confirmed that all strains catalysed the hydrolysis of nitriles by a nitrile hydratase/amidase system. Acetonitrile and benzonitrile, when used as growth substrates for enzyme induction experiments, had a significant influence on the biotransformation activities towards various nitriles and amides. The specific activities of selected deep-sea and terrestrial acetonitrile-grown bacteria against a suite of nitriles and amides were higher than those of the only other reported marine nitrile-hydrolysing R. erythropolis, isolated from a shallow sediment. The increase of nitrile chain length appeared to have negative influence on the nitrile hydratase activity of acetonitrile-grown bacteria, but the same was not true for benzonitrile-grown bacteria. The nitrile hydratases and amidases were constitutive in 10 of the 16 deep-sea and terrestrial actinomycetes studied, and one strain showed an inducible hydratase and a constitutive amidase. Most of the deep-sea strains had constitutive activities and showed some of the highest activities and broadest substrate specificities of organisms included in this study. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of dissolved aromatic hydrocarbons on the growth of marine bacteria in batch culture.

Dissolved aromatic hydrocarbons were found to decrease growth rate and maximum cell density of marine bacteria in batch cultures. The magnitude of the decrement was observed to be a function of concentration of the hydrocarbon and inherent toxicity. The inherent toxicity was observed to increase inversely with solubility such that naphthalene at 100 muM concentration demonstrated a toxic effect similar to benzopyrene at 0.02 muM. A partial oxidation product of naphthalene was found to be more effective in decreasing growth parameters than naphthalene at equivalent concentrations and to cause complete cessation of growth at the higher concentrations permitted by its polar structure.     

The effect of aromatic compounds on the work of activated sludge.

The resistance of bacteria occurring in activated sludge purifying refining-petrochemical wastewaters to phenol, cresol and pirocatechin as well as the possibility of the purification of synthetic wastewaters carrying high concentrations of these compounds by sludge composed of resistant strains was studied. The most toxic of the studied compounds was phenol. Six out of 29 strains were resistant to high concentration of phenol (1000 mg/l). Activated sludge synthesized from strains resistant to phenol, cresol and pyrocatechin was tolerant to high concentrations of these compounds. Worsening of the work of the sludge, expressed by decrease of GOD, was observed at the concentrations of phenol, cresol and pyrocatechin of 2000, 400 and 300 mg/l, respectively.     

Stimulation by Gangliosides of Viability of Rat Brain Neurons and of Neuronal PC12 Cell Line under Conditions of Oxidative Stress

We studied effect of gangliosides on viability of brain neurons and neuronal PC12 cell line exposed to toxic concentrations of compounds activating free radical reactions. It is found that preincubation of cerebellar granule cells and PC12 cells with micromolar concentrations of ganglioside GM1 increases statistically significantly viability of these cells submitted to inductors of oxidative stress, such as hydrogen peroxide and the Fe²⁺-ascorbate system However, the effect of ganglioside GM1 in the PC12 cells failed to be revealed 1–2 days after treatment of the cells with trypsin, which indicates an importance of interaction of gangliosides with surface proteins for realization of their protective action. GM1, GD1a, and other gangliosides were shown to produce the neuroprotective effect on cerebellar granule cells in the presence of toxic glutamate concentrations. Not only micro-, but also nanomolar concentrations of these gangliosides increased statistically significantly the neuronal viability, although at micromolar concentrations this effect as a rule was more pronounced. The obtained data allow suggesting that the neuroprotective action of gangliosides is determined to a considerable degree by their ability to inhibit free-radical reactions in nerve cells.