Aromatic compounds degradation plays a role in colonization of Arabidopsis thaliana and Acacia caven by Cupriavidus pinatubonensis JMP134

Plant rhizosphere and internal tissues may constitute a relevant habitat for soil bacteria displaying high catabolic versatility towards xenobiotic aromatic compounds. Root exudates contain various molecules that are structurally related to aromatic xenobiotics and have been shown to stimulate bacterial degradation of aromatic pollutants in the rhizosphere. The ability to degrade specific aromatic components of root exudates could thus provide versatile catabolic bacteria with an advantage for rhizosphere colonization and growth. In this work, Cupriavidus pinatubonensis JMP134, a well-known aromatic compound degrader (including the herbicide 2,4-dichlorophenoxyacetate, 2,4-D), was shown to stably colonize Arabidopsis thaliana and Acacia caven plants both at the rhizoplane and endorhizosphere levels and to use root exudates as a sole carbon and energy source. No deleterious effects were detected on these colonized plants. When a toxic concentration of 2,4-D was applied to colonized A. caven, a marked resistance was induced in the plant, showing that strain JMP134 was both metabolically active and potentially beneficial to its host. The role for the β-ketoadipate aromatic degradation pathway during plant root colonization by C. pinatubonensis JMP134 was investigated by gene inactivation. A C. pinatubonensis mutant derivative strain displayed a reduced ability to catabolise root exudates isolated from either plant host. In this mutant strain, a lower competence in the rhizosphere of A. caven was also shown, both in gnotobiotic in vitro cultures and in plant/soil microcosms.     

Functions of flavin reductase and quinone reductase in 2,4,6-trichlorophenol degradation by Cupriavidus necator JMP134

The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (2,4,6-TCP), a toxic pollutant. TcpA is a reduced flavin adenine dinucleotide (FADH₂)-dependent monooxygenase that converts 2,4,6-TCP to 6-chlorohydroxyquinone. It has been implied via genetic analysis that TcpX acts as an FAD reductase to supply TcpA with FADH₂, whereas the function of TcpB in 2,4,6-TCP degradation is still unclear. In order to provide direct biochemical evidence for the functions of TcpX and TcpB, the two corresponding genes (tcpX and tcpB) were cloned, overexpressed, and purified in Escherichia coli. TcpX was purified as a C-terminal His tag fusion (TcpX_H) and found to possess NADH:flavin oxidoreductase activity capable of reducing either FAD or flavin mononucleotide (FMN) with NADH as the reductant. TcpX_H had no activity toward NADPH or riboflavin. Coupling of TcpX_H and TcpA demonstrated that TcpX_H provided FADH₂ for TcpA catalysis. Among several substrates tested, TcpB showed the best activity for quinone reduction, with FMN or FAD as the cofactor and NADH as the reductant. TcpB could not replace TcpX_H in a coupled assay with TcpA for 2,4,6-TCP metabolism, but TcpB could enhance TcpA activity. Further, we showed that TcpB was more effective in reducing 6-chlorohydroxyquinone than chemical reduction alone, using a thiol conjugation assay to probe transitory accumulation of the quinone. Thus, TcpB was acting as a quinone reductase for 6-chlorohydroxyquinone reduction during 2,4,6-TCP degradation.     

Characterization of MnpC, a Hydroquinone Dioxygenase Likely Involved in the meta-Nitrophenol Degradation by Cupriavidus necator JMP134

Cupriavidus necator JMP134 utilizes meta-nitrophenol (MNP) as the sole source of carbon, nitrogen, and energy. The metabolic reconstruction of MNP degradation performed in silico suggested that MnpC might have played an important role in MNP degradation. In order to experimentally confirm the prediction, we have now characterized the mnpC-encoded (amino)hydroquinone dioxygenase involved in the ring-cleavage reaction of MNP degradation. Real-time PCR analysis indicated that mnpC played an essential role in MNP degradation. MnpC was purified to homogeneity as an N-terminal six-His-tagged fusion protein, and it was proved to be a dimer as demonstrated by gel filtration. MnpC was a Fe²⁺- and Mn²⁺-dependent dioxygenase, catalyzing the ring-cleavage of hydroquinone to 4-hydroxymuconic semialdehyde in vitro and proposed as an aminohydroquinone dioxygenase involved in MNP degradation in vivo. Phylogenetic analysis suggested that MnpC diverged from the other (chloro)hydroquinone dioxygenases at an earlier point, which might result in the preference for its physiological substrate.     

Differential Utilization of Carbon Substrates by Bacteria and Fungi in Tundra Soil

Little is known about the contribution of bacteria and fungi to decomposition of different carbon compounds in arctic soils, which are an important carbon store and possibly vulnerable to climate warming. Soil samples from a subarctic tundra heath were incubated with ¹³C-labeled glucose, acetic acid, glycine, starch, and vanillin, and the incorporation of ¹³C into different phospholipid fatty acids (PLFA; indicative of growth) and neutral lipid fatty acids (NLFA; indicative of fungal storage) was measured after 1 and 7 days. The use of ¹³C-labeled substrates allowed the addition of substrates at concentrations low enough not to affect the total amount of PLFA. The label of glucose and acetic acid was rapidly incorporated into the PLFA in a pattern largely corresponding to the fatty acid concentration profile, while glycine and especially starch were mainly taken up by bacteria and not fungi, showing that different groups of the microbial community were responsible for substrate utilization. The ¹³C-incorporation from the complex substrates (starch and vanillin) increased over time. There was significant allocation of ¹³C into the fungal NLFA, except for starch. For glucose, acetic acid, and glycine, the allocation decreased over time, indicating use of the storage products, whereas for vanillin incorporation into fungal NLFA increased during the incubation. In addition to providing information on functioning of the microbial communities in an arctic soil, our study showed that the combination of PLFA and NLFA analyses yields additional information on the dynamics of substrate degradation.Bacteria and fungi comprise more than 90% of the soil microbial biomass and are the main agents for decomposition of organic matter in soil. Until recently it was thought that these two organism groups could be lumped together in this respect, and total microbial biomass or total activity (respiration) was often the only variable included in soil microbiology studies of decomposition and soil organic matter turnover (39). However, there is increasing evidence suggesting that whether decomposition is performed by bacteria or fungi, thereby channeling energy through the bacterial or the fungal food web, has profound effects on the ecosystem. Such effects can have direct influence on the higher trophic levels in the food web (30) or indirect effects on nutrient mineralization rates (14) and nutrient transfer (19, 20), and they can even determine the extent of carbon sequestration in the soil (37). The situation becomes even more complex when the impact of changes in climate, nitrogen availability, and litter input on the balance between bacteria and fungi is taken into account. The Arctic region has been identified as an area that will be especially vulnerable to these changes (3).Little is known about the contribution of bacteria and fungi to the utilization of plant-derived carbon substrates in arctic soils. Differentiation of the bacterial and fungal contributions to decomposition has hitherto relied to a large extent on changes in bacterial and fungal biomasses, for example, by analysis of patterns of phospholipid fatty acids (PLFA) (40). PLFA are components of the cell membrane, and some of the PLFA extracted from the soil are characteristic for a certain microbial group in the environment. However, for changes in PLFA concentrations after the addition of substrates to be detected, substrates often have to be added at unrealistically large amounts. Even then only small changes in the PLFA concentrations will often be detected (35).One way of overcoming these problems is to follow the incorporation of ¹³C label from added substrates into specific fatty acids (8, 17). This approach adds a new dimension—metabolic function—to the study of soil microbial communities without the need of cultivation. It also increases the sensitivity in tracing responses of organism groups to different substrates as the addition of substrates at low and more realistic concentrations with high specific ¹³C label will induce large changes in the ¹³C concentration of the PLFA without changing the total amount of PLFA.Carbon-13 labeling has been used to follow uptake of recent photosynthates (11, 13, 27), pure substrates (10, 12, 32, 33, 41), and complex labeled plant material (28, 41, 43, 44) into PLFA although seldom in arctic soils. However, microorganisms incorporate carbon not only into phospholipids (indicating growth) but also into storage products, for example, when a nutrient other than carbon is limiting growth or under growth-restricting conditions. Thus, with excess carbon both bacteria and fungi will store carbon for later need, for example, as polyhydroxyalkanoate or glycogen (bacteria) and triacylglycerols (fungi). Thus, neutral lipid fatty acids (NLFA) of fungal origin can be used to indicate storage in fungi (4). Degraded PLFA, resulting in diacylglycerols, will also end up in the corresponding NLFA fraction, and NLFA has thus been suggested as an indicator of recently dead bacterial biomass (42). Therefore, the NLFA/PLFA ratio serves two purposes: for fungal lipids a higher NLFA/PLFA ratio would indicate allocation of lipids to energy storage while for bacterial lipids it would indicate turnover of this bacterial group. However, the latter will probably be of minor importance during short incubations. As far as we know, no studies on soil microorganisms have used incorporation of ¹³C from substrates to indicate both effects on growth (incorporation into PLFA) and storage (incorporation into NLFA).We assessed the uptake of ¹³C-labeled substrates into lipid biomarkers of different microbial groups in a laboratory incubation experiment using soil from an arctic tundra heath. The selected substrates represented carbon sources present in soil. Glucose, acetic acid, and glycine are simple compounds common in plant root exudates, and glycine is also a nitrogen source. Starch is a very common polysaccharide in plant residues. Vanillin is a common product of lignin depolymerization (18) containing a phenol ring and is often used as a model substance to indicate lignin degradation. Starch and vanillin are therefore examples of more complex substrates and are supposedly more difficult to decompose. We followed the incorporation of the label into different PLFA and NLFA over time. We hypothesized that ¹³C from the simple compounds would be more rapidly incorporated into microbial PLFA than ¹³C from the more complex substrates (more rapid growth), and thus we expected ¹³C emanating from the complex substrates to increase in concentration in the PLFA and NLFA over time. We also hypothesized that bacteria would be better than fungi in utilizing simple compounds while the label from the more complex substrates would preferentially be incorporated into PLFA, indicating fungi (6, 29). We also expected ¹³C from the C-rich substrates to be incorporated into NLFA (fungal storage) to a larger extent than C from glycine, which also serves as a nitrogen source (4). However, with time the carbon in storage structures would decrease as it would be used for growth or maintenance energy.     

Use of 13C Nuclear Magnetic Resonance To Assess Fossil Fuel Biodegradation: Fate of [1-13C]Acenaphthene in Creosote Polycyclic Aromatic Compound Mixtures Degraded by Bacteria

[1-¹³C]acenaphthene, a tracer compound with a nuclear magnetic resonance (NMR)-active nucleus at the C-1 position, has been employed in conjunction with a standard broad-band-decoupled ¹³C-NMR spectroscopy technique to study the biodegradation of acenaphthene by various bacterial cultures degrading aromatic hydrocarbons of creosote. Site-specific labeling at the benzylic position of acenaphthene allows ¹³C-NMR detection of chemical changes due to initial oxidations catalyzed by bacterial enzymes of aromatic hydrocarbon catabolism. Biodegradation of [1-¹³C]acenaphthene in the presence of naphthalene or creosote polycyclic aromatic compounds (PACs) was examined with an undefined mixed bacterial culture (established by enrichment on creosote PACs) and with isolates of individual naphthalene- and phenanthrene-degrading strains from this culture. From ¹³C-NMR spectra of extractable materials obtained in time course biodegradation experiments under optimized conditions, a number of signals were assigned to accumulated products such as 1-acenaphthenol, 1-acenaphthenone, acenaphthene-1,2-diol and naphthalene 1,8-dicarboxylic acid, formed by benzylic oxidation of acenaphthene and subsequent reactions. Limited degradation of acenaphthene could be attributed to its oxidation by naphthalene 1,2-dioxygenase or related dioxygenases, indicative of certain limitations of the undefined mixed culture with respect to acenaphthene catabolism. Coinoculation of the mixed culture with cells of acenaphthene-grown strain Pseudomonas sp. strain A2279 mitigated the accumulation of partial transformation products and resulted in more complete degradation of acenaphthene. This study demonstrates the value of the stable isotope labeling approach and its ability to reveal incomplete mineralization even when as little as 2 to 3% of the substrate is incompletely oxidized, yielding products of partial transformation. The approach outlined may prove useful in assessing bioremediation performance.Microbial degradation of polycyclic aromatic compounds (PACs) is increasingly being considered for bioremediation applications and has been proposed as an attractive approach to remediation technologies dealing with fossil fuel wastes (10, 11). However, biodegradation of PACs of creosote and of related hydrocarbons of petroleum by defined and undefined mixed bacterial cultures may result in elevated rates of formation and even accumulation of organic end products (3, 5, 20). Some of these products are toxic to certain test organisms (1, 3, 20). Such information may be relevant in determining, for example, why little change in toxicity is observed when creosote undergoes biodegradation in groundwater (11). Therefore, chemical and toxicological characterization of any biodegradation process is required before the method is applied.A realistic assessment of biodegradation efficacy requires delineation of a pollutant’s fate and effects beyond its depletion, as revealed by sensitive and exact analytical methods. For complex mixtures of chemicals, such as those found in coal- and petroleum-derived materials, this is not so readily accomplished. Assessment of efficacy cannot be based solely on rates of mineralization inferred from data obtained by trapping CO₂ released after the addition of a radioactively labeled tracer compound. Although this method is a convenient laboratory indicator of limited aspects of mineralization, it fails to provide information about the presence, nature, and distribution of organic end products or their interactions with soil and sedimentary matter.¹³C-nuclear magnetic resonance (NMR) spectroscopy, combined with ¹³C labeling, however, offers an approach whereby details of the chemical structures of individual components of PAC mixtures undergoing biodegradation can be investigated. With the ¹³C-labeled tracers available, ¹³C-NMR has been successfully applied to an investigation of the interaction of 2,4-dichlorophenol with humic matrixes (8) and to a study of the alteration of jet fuels undergoing thermal stress (9). For a more complete study of its utility, various specifically ¹³C-labeled constituents of fossil fuels are required for evaluation of this technique in assessing the biodegradation of coal- and petroleum-derived wastes.The applicability of this approach to the biodegradation of hydrocarbon mixtures is examined in the present study by using synthetic [1-¹³C]acenaphthene (99% ¹³C) introduced as a tracer compound into creosote PAC mixtures degraded by different undefined, mixed, and axenic bacterial cultures. The initial choice of acenaphthene labeled with ¹³C at a benzylic carbon as a tracer is based on the abundance of acenaphthene in creosote and the straightforward route of its synthesis. It also represents an extension of published work on acenaphthene oxidation catalyzed by naphthalene dioxygenase (16), biotransformation reactions catalyzed by bacteria and fungi (12, 14), and catabolism of acenaphthene by certain soil bacteria (19), with the consequent availability of suitable acenaphthene-utilizing microbial cultures.(Preliminary accounts of aspects of this work have appeared elsewhere [2, 17, 18].)     

Use of 13C Nuclear Magnetic Resonance To Assess Fossil Fuel Biodegradation: Fate of [1-13C]Acenaphthene in Creosote Polycyclic Aromatic Compound Mixtures Degraded by Bacteria

[1-¹³C]acenaphthene, a tracer compound with a nuclear magnetic resonance (NMR)-active nucleus at the C-1 position, has been employed in conjunction with a standard broad-band-decoupled ¹³C-NMR spectroscopy technique to study the biodegradation of acenaphthene by various bacterial cultures degrading aromatic hydrocarbons of creosote. Site-specific labeling at the benzylic position of acenaphthene allows ¹³C-NMR detection of chemical changes due to initial oxidations catalyzed by bacterial enzymes of aromatic hydrocarbon catabolism. Biodegradation of [1-¹³C]acenaphthene in the presence of naphthalene or creosote polycyclic aromatic compounds (PACs) was examined with an undefined mixed bacterial culture (established by enrichment on creosote PACs) and with isolates of individual naphthalene- and phenanthrene-degrading strains from this culture. From ¹³C-NMR spectra of extractable materials obtained in time course biodegradation experiments under optimized conditions, a number of signals were assigned to accumulated products such as 1-acenaphthenol, 1-acenaphthenone, acenaphthene-1,2-diol and naphthalene 1,8-dicarboxylic acid, formed by benzylic oxidation of acenaphthene and subsequent reactions. Limited degradation of acenaphthene could be attributed to its oxidation by naphthalene 1,2-dioxygenase or related dioxygenases, indicative of certain limitations of the undefined mixed culture with respect to acenaphthene catabolism. Coinoculation of the mixed culture with cells of acenaphthene-grown strain Pseudomonas sp. strain A2279 mitigated the accumulation of partial transformation products and resulted in more complete degradation of acenaphthene. This study demonstrates the value of the stable isotope labeling approach and its ability to reveal incomplete mineralization even when as little as 2 to 3% of the substrate is incompletely oxidized, yielding products of partial transformation. The approach outlined may prove useful in assessing bioremediation performance.     

Sugarcane Molasses: A Cheap Carbon Source for Calcite Production in Different Class of Soils using Stimulation of Indigenous Urease-producing Bacteria

Abstract

In this research, we investigated the abilities of three different concentration of sugarcane molasses as a carbon source to stimulate indigenous bacterial growth in different classes of soil, namely poorly graded sand (SP), silty sand (SM), and clayey sand (SC) (according to the Unified classification system). A total of 7, 10, and 15 days after the treatment, direct shear tests were performed on the untreated and treated samples. The calcite content on all direct shear samples was determined to further correlate it with the strength gains in the treated samples. The scanning electron microscopy (SEM) images, EDX analysis, and X-ray diffraction (XRD) patterns were taken before and after treatment for all samples to analyze the microbial-induced calcite precipitation (MICP) process. The SP soil samples showed the highest strength gains and also highest calcite content as compared with other two soil type. The peak cohesion intercept for SP-treated samples increased by 2.7–5.5 times as compared to the untreated samples for molasses concentration of 1–3?g/L, respectively. The treated samples became more dilative with the increase in molasses concentration. The sample with highest molasses concentration showed stiffer behavior in shear than the samples with lower concentration.     

Remarkable Recovery and Colonization Behaviour of Methane Oxidizing Bacteria in Soil After Disturbance Is Controlled by Methane Source Only

Little is understood about the relationship between microbial assemblage history, the composition and function of specific functional guilds and the ecosystem functions they provide. To learn more about this relationship we used methane oxidizing bacteria (MOB) as model organisms and performed soil microcosm experiments comprised of identical soil substrates, hosting distinct overall microbial diversities (i.e., full, reduced and zero total microbial and MOB diversities). After inoculation with undisturbed soil, the recovery of MOB activity, MOB diversity and total bacterial diversity were followed over 3 months by methane oxidation potential measurements and analyses targeting pmoA and 16S rRNA genes. Measurement of methane oxidation potential demonstrated different recovery rates across the different treatments. Despite different starting microbial diversities, the recovery and succession of the MOB communities followed a similar pattern across the different treatment microcosms. In this study we found that edaphic parameters were the dominant factor shaping microbial communities over time and that the starting microbial community played only a minor role in shaping MOB microbial community     

Utilization of DNA as a Sole Source of Phosphorus, Carbon, and Energy by Shewanella spp.: Ecological and Physiological Implications for Dissimilatory Metal Reduction

The solubility of orthophosphate (PO₄³⁻) in iron-rich sediments can be exceedingly low, limiting the bioavailability of this essential nutrient to microbial populations that catalyze critical biogeochemical reactions. Here we demonstrate that dissolved extracellular DNA can serve as a sole source of phosphorus, as well as carbon and energy, for metal-reducing bacteria of the genus Shewanella. Shewanella oneidensis MR-1, Shewanella putrefaciens CN32, and Shewanella sp. strain W3-18-1 all grew with DNA but displayed different growth rates. W3-18-1 exhibited the highest growth rate with DNA. While strain W3-18-1 displayed Ca²⁺-independent DNA utilization, both CN32 and MR-1 required millimolar concentrations of Ca²⁺ for growth with DNA. For S. oneidensis MR-1, the utilization of DNA as a sole source of phosphorus is linked to the activities of extracellular phosphatase(s) and a Ca²⁺-dependent nuclease(s), which are regulated by phosphorus availability. Mass spectrometry analysis of the extracellular proteome of MR-1 identified one putative endonuclease (SO1844), a predicted UshA (bifunctional UDP-sugar hydrolase/5′ nucleotidase), a predicted PhoX (calcium-activated alkaline phosphatase), and a predicted CpdB (bifunctional 2′,3′ cyclic nucleotide 2′ phosphodiesterase/3′ nucleotidase), all of which could play important roles in the extracellular degradation of DNA under phosphorus-limiting conditions. Overall, the results of this study suggest that the ability to use exogenous DNA as the sole source of phosphorus is widespread among the shewanellae, and perhaps among all prokaryotes, and may be especially important for nutrient cycling in metal-reducing environments.     

Volatile Ketone Formation in Bacteria: Release of 3-Oxopentanoate by Soil Pseudomonads During Growth on Heptanoate

During enrichments to search for soil bacteria that form the volatile ketones, acetone and butanone, we isolated pure cultures of Pseudomonas aureofaciens, P. fluorescens, and P. putida that excrete the β-keto-acid, 3-oxopentanoate (3-OPA), when grown on heptanoic acid as carbon source. Analysis of 3-OPA used enzymatic decarboxylation by acetoacetate decarboxylase to yield butanone, which was detected by headspace gas chromatography. The formation of 3-OPA was strongly dependent on heptanoic acid concentration, the level of oxygen, and the state of growth, and was not seen with even-chain or other odd-chain fatty acids. Uptake of 3-OPA during stationary phase of growth is probably related to polyhydroxyalkanoate (PHA) formation in these isolates. A model for formation and release of 3-OPA is proposed. Received: 28 August 2000 / Accepted: 2 October 2000     

Effects of Grazing by Flagellates on Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Soil Columns

The enhanced mineralization of immobilized nitrogen by bacteriophagous protozoa has been thought to favor the nitrification process in soils in which nitrifying bacteria must compete with heterotrophic bacteria for the available ammonium. To obtain more insight into this process, the influence of grazing by the flagellate Adriamonas peritocrescens on the competition for ammonium between the chemolithotrophic species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis in the presence of Nitrobacter winogradskyi was studied in soil columns, which were continuously percolated with media containing 5 mM ammonium and different amounts of glucose at a dilution rate of 0.007 h^-1 (liquid volumes). A. globiformis won the competition for ammonium. The grazing activities of the flagellates had two prominent effects on the competition between N. europaea and A. globiformis. First, the distribution of ammonium over the profile of the soil columns was more uniform in the presence of flagellates than in their absence. In the absence of flagellates, relatively high amounts of ammonium accumulated in the upper layer (0 to 3 cm), whereas in the underlying layers the ammonium concentrations were low. In the presence of flagellates, however, considerable amounts of ammonium were found in the lower layers, whereas less ammonium accumulated in the upper layer. Second, the potential ammonium-oxidizing activity of N. europaea was stimulated in the presence of flagellates. The numbers of N. europaea at different glucose concentrations in the presence of flagellates were comparable to those in the absence of protozoa. However, in the presence of flagellates, the potential ammonium-oxidizing activities were four to five times greater than those in the absence of protozoa.     

Iron Reduction and Soil Phosphorus Solubilization in Humid Tropical Forests Soils: The Roles of Labile Carbon Pools and an Electron Shuttle Compound

The affinity of iron oxides and hydroxides for phosphorus is thought to contribute to phosphorus limitation to net primary productivity in humid tropical forests on acidic, highly weathered soils. Perennially warm, humid conditions and high biological activity in these soils can result in fluctuating redox potential that in turn leads to considerable iron reduction in the presence of labile carbon and humic substances. We investigated the effects of reducing conditions in combination with the addition of labile carbon substrates (glucose and acetate) and an electron shuttle compound on iron reduction and phosphorus release in a humid tropical forest soil. Glucose or acetate was added to soils as a single dose at the beginning of the experiment, and as pulsed inputs over time, which more closely mimics patterns in labile carbon availability. Iron reduction and phosphorus mobilization were weakly stimulated by a single low level addition of carbon, and the addition of the electron shuttle compound with or without added carbon. Pulsed labile carbon additions produced a significant increase in soil pH, soluble iron, and phosphorus concentrations. Pulsed labile carbon inputs also promoted the precipitation of ferrous hydroxide complexes which could increase the capacity for P sorption, although our results suggest that rates of P solubilization exceeded re-adsorption. Plant and microbial P demand are also likely to serve as an important sinks for released P, limiting the role of P re-adsorption. Our results suggest that reducing conditions coupled with periodic carbon inputs can stimulate iron reduction and a corresponding increase in soil phosphorus mobilization, which may provide a source of phosphorus to plants and microorganisms previously undocumented in these ecosystems.     

Detection of Plasmid Transfer from Pseudomonas fluorescens to Indigenous Bacteria in Soil by Using Bacteriophage phiR2f for Donor Counterselection

The transfer of a genetically marked derivative of plasmid RP4, RP4p, from Pseudomonas fluorescens to members of the indigenous microflora of the wheat rhizosphere was studied by using a bacteriophage that specifically lyses the donor strain and a specific eukaryotic marker on the plasmid. Transfer of RP4p to the wheat rhizosphere microflora was observed, and the number of transconjugants detected was approximately 10 transconjugants per g of soil when 10 donor cells per g of soil were added; transfer in the corresponding bulk soil was slightly above the limit of detection. All of the indigenous transconjugants which we analyzed contained a 60-kb plasmid and were able to transfer this plasmid to a Nx RpP. fluorescens recipient strain. The indigenous transconjugants were identified as belonging to Pseudomonas spp., Enterobacter spp., Comamonas spp., and Alcaligenes spp.     

Spatial and Temporal Variability in the Amount and Source of Dissolved Organic Carbon: Implications for Ultraviolet Exposure in Amphibian Habitats

The amount, chemical composition, and source of dissolved organic carbon (DOC), together with in situ ultraviolet (UV-B) attenuation, were measured at 1–2 week intervals throughout the summers of 1999, 2000, and 2001 at four sites in Rocky Mountain National Park (Colorado). Eight additional sites, four in Sequoia and Kings Canyon National Park/John Muir Wilderness (California) and four in Glacier National Park (Montana), were sampled during the summer of 2000. Attenuation of UV-B was significantly related to DOC concentrations over the three years in Rocky Mountain (R² = 0.39, F = 25.71, P < 0.0001) and across all parks in 2000 (R² = 0.44, F = 38.25, P < 0.0001). The relatively low R² values, however, reflect significant temporal and spatial variability in the specific attenuation per unit DOC. Fluorescence analysis of the fulvic acid DOC fraction (roughly 600–2,000 Daltons) indicated that the source of DOC significantly affected the attenuation of UV-B. Sites in Sequoia–Kings Canyon were characterized by DOC derived primarily from algal sources and showed much deeper UV-B penetration, whereas sites in Glacier and Rocky Mountain contained a mix of algal and terrestrial DOC-dominated sites, with more terrestrially dominated sites characterized by greater UV-B attenuation per unit DOC. In general, site characteristics that promoted the accumulation of terrestrially derived DOC showed greater attenuation of UV-B per unit DOC; however, catchment vegetation and soil characteristics, precipitation, and local hydrology interacted to make it difficult to predict potential exposure from DOC concentrations.     

Identification of a Novel Metabolite in the Degradation of Pyrene by Mycobacterium sp. Strain AP1: Actions of the Isolate on Two- and Three-Ring Polycyclic Aromatic Hydrocarbons

Mycobacterium sp. strain AP1 grew with pyrene as a sole source of carbon and energy. The identification of metabolites accumulating during growth suggests that this strain initiates its attack on pyrene by either monooxygenation or dioxygenation at its C-4, C-5 positions to give trans- or cis-4,5-dihydroxy-4,5-dihydropyrene, respectively. Dehydrogenation of the latter, ortho cleavage of the resulting diol to form phenanthrene 4,5-dicarboxylic acid, and subsequent decarboxylation to phenanthrene 4-carboxylic acid lead to degradation of the phenanthrene 4-carboxylic acid via phthalate. A novel metabolite identified as 6,6′-dihydroxy-2,2′-biphenyl dicarboxylic acid demonstrates a new branch in the pathway that involves the cleavage of both central rings of pyrene. In addition to pyrene, strain AP1 utilized hexadecane, phenanthrene, and fluoranthene for growth. Pyrene-grown cells oxidized the methylenic groups of fluorene and acenaphthene and catalyzed the dihydroxylation and ortho cleavage of one of the rings of naphthalene and phenanthrene to give 2-carboxycinnamic and diphenic acids, respectively. The catabolic versatility of strain AP1 and its use of ortho cleavage mechanisms during the degradation of polycyclic aromatic hydrocarbons (PAHs) give new insight into the role that pyrene-degrading bacterial strains may play in the environmental fate of PAH mixtures.     

Preference for plants damaged by conspecific larvae in ovipositing cabbage root flies: influence of stimuli from leaf surface and roots

In laboratory dual-choice assays females of the cabbage root fly, Delia radicum, prefer for oviposition plants with roots damaged by conspecific larvae to undamaged controls. Cauliflower and kale plants were inoculated with root fly eggs (25 per plant) and the hatching larvae were allowed to feed on the roots for various periods of time (1–17 days). After 4 (cauliflower) or 5 (kale) days of larval feeding the oviposition preference was most pronounced and flies laid between 64% and 68% of their eggs near plants with damaged roots. Later, with increasing damage but fewer surviving, and thus actively feeding, larvae, the magnitude of the preference declined. The preference for plants already damaged by conspecific larvae may contribute to the previously observed aggregated distribution of D. radicum eggs in Brassica crop fields.Further experiments revealed that the sensory cues inducing this oviposition preference originate from the complex consisting of the damaged roots, the surrounding substrate (soil) and associated microbes, rather than from the aerial plant parts. In choice assays using the root-substrate complex of damaged and control plants (aerial parts removed), the observed preference for damaged roots was similar to that found for the entire plant but was more pronounced. The damaged roots alone, compared to control roots, received up to 72% (cauliflower) and 75% (kale) of the eggs. By contrast, surrogate leaves sprayed with methanolic leaf surface extracts from the most preferred plants which had been damaged were not discriminated from surrogate leaved sprayed with extracts of the respective control plants. Analysis of glucosinolate levels in methanolic leaf surface extracts revealed that root damage resulted in enhanced concentrations of indole-glucosinolates on the leaf surface in kale but not in cauliflower. Although indole-glucosinolates are oviposition stimulants for the cabbage root fly, the induced changes were apparently too small to influence oviposition behaviour.     

Detection of Plasmid Transfer from Pseudomonas fluorescens to Indigenous Bacteria in Soil by Using Bacteriophage φR2f for Donor Counterselection

The transfer of a genetically marked derivative of plasmid RP4, RP4p, from Pseudomonas fluorescens to members of the indigenous microflora of the wheat rhizosphere was studied by using a bacteriophage that specifically lyses the donor strain and a specific eukaryotic marker on the plasmid. Transfer of RP4p to the wheat rhizosphere microflora was observed, and the number of transconjugants detected was approximately 10³ transconjugants per g of soil when 10⁷ donor cells per g of soil were added; transfer in the corresponding bulk soil was slightly above the limit of detection. All of the indigenous transconjugants which we analyzed contained a 60-kb plasmid and were able to transfer this plasmid to a Nx^r Rp^rP. fluorescens recipient strain. The indigenous transconjugants were identified as belonging to Pseudomonas spp., Enterobacter spp., Comamonas spp., and Alcaligenes spp.