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PubMed2003

Experimental evaluation of the processes resulting from the introduction of the transgenic microorganism Escherichia coli Z905/pPHL7 (lux+) into aquatic microcosms.

The processes resulting from the introduction of the tranagenic microorganism (TM) E. coli Z905/pPHL7 into aquatic microcosms have been modeled experimentally. It has been shown that the TM E. coli is able to adapt to a long co-existence with indigenous heterotrophic microflora in variously structured microcosms. In more complex microcosms the numerical dynamics of the introduced E. coli Z905/pPHL7 population is more stable. In the TM populations staying in the microcosms for a prolonged time, changes are recorded in the phenotypic expression of plasmid genes (ampicillin resistance and the luminescence level) and chromosome genes (morphological and physiological traits). However, in our study microcosms, the recombinant plasmid persisted in the TM cells for 6 years after the introduction, and as the population adapts to the conditions of the microcosms, the efficiency of the cloned gene expression in the cells is restored. In the microcosms with high microalgal counts (10(7) cells/ml), cells with a high threshold of sensitivity to ampicillin dominate in the population of the TM E. coli Z905/pPHL7.

Adaptation, Physiological↗

PubMed2026

Carbon monoxide-driven proton respiration enables facultative anaerobes to survive electron acceptor limitation.

Diverse microorganisms couple the oxidation of carbon monoxide gas (CO) to the reduction of protons, producing hydrogen gas (H2) using nickel-containing CO dehydrogenase/energy-converting hydrogenase (Ni-CODH/ECH). Although this process yields one of the lowest free-energy gains in biology, its physiological role at environmentally relevant CO levels remains unresolved. Here, we show that Ni-CODH/ECH functions as a survival-oriented energy conservation system that enables heterotrophic facultative anaerobes to survive electron acceptor limitation, rather than primarily supporting growth or CO detoxification. Analysis of 387 genomes of Anoxybacillaceae species revealed that Ni-CODH/ECH had a patchy distribution and, with one exception, was mutually exclusive with the oxygen-tolerant molybdenum-containing CODH, suggesting ecological specialization. Culture experiments using three isolates (Parageobacillus sp. G301, P. thermoglucosidasius NBRC 107763, and Thermolongibacillus altinsuensis B1-1) demonstrated that CO-dependent proton respiration is activated during stationary phase when exogenous electron acceptors are limiting, maintaining cell density under 25% CO, whereas no effect was observed in a Ni-CODH knockout (ΔcooCSF) strain. RNA-seq analysis of Parageobacillus sp. G301 under twelve conditions revealed that Ni-CODH/ECH genes are highly expressed (top 0.2%-1.9% of all genes) under electron acceptor-free conditions, independent of CO presence, under the predicted control of the redox-dependent transcriptional repressor Rex. ΔcooCSF cultures accumulated more CO than the wild-type (WT), suggesting trace CO scavenging by the WT. Together, our results redefine Ni-CODH/ECH as a redox-regulated auxiliary energy-conservation strategy that supports survival and maintenance in anaerobic energy-limited environments using two ubiquitous substrates. This work extends the carboxydovore paradigm of trace gas-based survival from aerobic to spatiotemporally variable anaerobic environments.

Carbon Monoxide↗

PubMed2001

Fundamental processes within natural and constructed wetland ecosystems: short-term versus long-term objectives.

Use of wetland ecosystems for water pollution control consists essentially of sustained induced disturbances as pollutants are loaded to complex biological communities. Objectives are to maximize pollutant loading, incorporation, and retention while maintaining highest levels of community metabolism and minimal alteration of community structure. Several basic processes are emphasized: (a) macrophyte productivity in relation to shoot:root ratios, and nutrient availability; (b) macrophyte life history strategies, succession, and biodiversity under constant pollutant stress; (c) importance of standing dead and particulate detritus; (d) functions and controlling mechanisms of heterotrophic and autotrophic periphyton in pollutant retention and recycling; (e) coupling of microbial metabolism to macrophyte retention of pollutants; (f) gaseous losses to the atmosphere; (g) losses of dissolved organic matter and its utilization; and (h) water losses by evapotranspiration and effects on wetland efficacy. Short-term wetland removal efficiencies are confounded by massive variations in retentive capacities diurnally, seasonally, and spatially, in exceeding physiological tolerance levels, and in species succession. Problems of channelization, altered microhydrology, and assimilation/retention are major in natural and non-engineered ecosystems. Wetlands are highly ephemeral and variable in their capabilities for sequestering and retention of nutrients and other pollutants.

Ecosystem↗

PubMed2005

The HiPIP from the acidophilic Acidithiobacillus ferrooxidans is correctly processed and translocated in Escherichia coli, in spite of the periplasm pH difference between these two micro-organisms.

The gene encoding a putative high-potential iron-sulfur protein (HiPIP) from the strictly acidophilic and chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 33020 has been cloned and sequenced. This potential HiPIP was overproduced in the periplasm of the neutrophile and heterotroph Escherichia coli. As shown by optical and EPR spectra and by electrochemical studies, the recombinant protein has all the biochemical properties of a HiPIP, indicating that the iron-sulfur cluster was correctly inserted. Translocation of this protein in the periplasm of E. coli was not detected in a DeltatatC mutant, indicating that it is dependent on the Tat system. The genetic organization of the iro locus in strains ATCC 23270 and ATCC 33020 is different from that found in strains Fe-1 and BRGM. Indeed, in A. ferrooxidans ATCC 33020 and ATCC 23270 (the type strain), iro was not located downstream from purA but was instead downstream from petC2, encoding cytochrome c1 from the second A. ferrooxidans cytochrome bc1 complex. These findings underline the genotypic heterogeneity within the A. ferrooxidans species. The results suggest that Iro transfers electrons from a cytochrome bc1 complex to a terminal oxidase, as proposed for the HiPIP in photosynthetic bacteria.

Acidithiobacillus↗

PubMed2003

Biogeochemical processes and microbial characteristics across groundwater-surface water boundaries of the Hanford Reach of the Columbia River.

Biogeochemical processes within riverbed hyporheic zones (HZ) can potentially impact the fate and transport of contaminants. We evaluated a modified freeze core technique for the collection of intact cobble-bed samples from the Columbia River HZ along a stretch of the Hanford Reach in Washington State and investigated microbiological and geochemical parameters of corresponding frozen and unfrozen samples. During three sampling periods (March, May, and November 2000), relatively high numbers of viable aerobic heterotrophic bacteria were recovered from both unfrozen (10(6)-10(7) cfu/g) and frozen samples (10(5)-10(6) cfu/g). Relatively large populations of sulfate-, nitrate-, and iron-reducing bacteria were present, and significant concentrations of acid-volatile sulfide were measured in some samples, indicating that anoxic regions exist within this zone. Cr(VI), a priority groundwater pollutant on adjacent U.S. Department of Energy lands, was probably removed from solution in HZ samples by a combination of microbial activity and chemical reduction, presumably via products of anaerobic microbial metabolism. These results suggest that biogeochemical processes in the Columbia River HZ may contribute to the natural attenuation of Cr(VI). Although freezing modestly diminished recovery of viable bacteria, freeze core techniques proved reliable for the collection of intact hyporheic sediments.

Biodegradation, Environmental↗

PubMed1989

A point mutation in the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase affects holoenzyme assembly in Nicotiana tabacum.

In photosynthetic eukaryotes, the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is composed of eight large and eight small subunits. Chloroplast-coded large subunits are found in association with chaperonins (binding proteins) of 60-61 kd to form a high mol. wt pre-assembly complex (B-complex). We have isolated a heterotrophic, maternally-inherited mutant from Nicotiana tabacum var. Xanthi which accumulates the B-complex but contains no Rubisco holoenzyme. The B-complex of the mutant dissociates in the presence of ATP, as does that of the wild-type. Processing of the nuclear-coded small subunit takes place in the mutant and neither large nor small subunits accumulate. The large subunit gene from mutant and wild-type plants was cloned and sequenced. A single nucleotide difference was found between them predicting an amino acid change of serine to phenylalanine at position 112 in the mutant. Based on the resolved structure of N.tabacum Rubisco, it is argued that the alteration at position 112 prevents holoenzyme assembly by interfering with large subunit assembly.

Amino Acid Sequence↗

PubMed2002

Diatom cultivation and biotechnologically relevant products. Part I: cultivation at various scales.

Biotechnological applications of diatoms are still in development. Further development at the industrial scale will depend on optimisation of the culture process with the aim of reducing costs. Because of the photoautotrophic status of the majority of diatoms, microalgal cultures suffer from the limitation of light diffusion, which requires the development of suitable photobioreactors. Thus, genetically engineered microalgae that may be cultivated in heterotrophic conditions present a new opportunity. Other limiting factors, such as nutrients (phosphate, nitrogen, silicon), pH, temperature, bioturbation and many more must be taken into account. Most of the time, metabolic stress conditions lead to an overproduction of the products of interest, with a decrease in biomass production as a consequence. Outdoor cultures in open ponds are usually devoted to aquaculture for the feeding of shrimps and bivalve molluscs (commercial production), while closed axenic indoor/outdoor photobioreactors are used for biotechnological compounds of homogeneous composition (still at the laboratory scale). In addition to the optimum culture conditions that have to be taken into account for photobioreactor design, the localisation of produced metabolites (intra- or extracellular) may also be taken into account when choosing the design. Microalgal cell immobilisation may be a suitable technique for application to benthic diatoms, which are usually sensitive to bioturbation and/or metabolites which may be overexpressed.

Animal Feed↗

PubMed2005

Effects of antibacterials use in aquaculture on biogeochemical processes in marine sediment.

The effects of chloramphenicol on microorganism in marine sediment were studied by spiked experiments in this paper. The results showed that high concentrations of chloramphenicol could inhibit the activities of microorganism in sediment, and that the growth of strains Pseudomons and Acinetobacter in sediment that can degrade organic matters were inhibited apparently. Furthermore, the resistance of heterotrophic bacteria in sediment had developed due to the use of antibacterials. Based on the above results potential environmental effects of antibacterials on microorganism in marine sediment were analyzed.

Acinetobacter↗

PubMed2002

Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is post-translationally phosphorylated in heterotrophic cells of wheat (Triticum aestivum).

In wheat, non-phosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) was found to be encoded by one gene giving rise to a single protein. However, Western blots revealed two different subunits of about 58 and 60 kDa in endosperm and shoots. The latter was attributed to in vivo phosphorylation of shoot GAPN. No modification occurred in leaves, where the enzyme is composed by a single 58 kDa polypeptide. GAPN partially purified from shoots and endosperm was dephosphorylated in vitro with alkaline phosphatase. Phosphorylated GAPN exhibited similar affinity for substrates but a lower V(max) compared to the non-phosphorylated enzyme. Results suggest that reversible phosphorylation of GAPN could regulate NADPH production in the cytosol of heterotrophic plant cells.

Amino Acid Sequence↗

PubMed2002

Active biomass in activated sludge mixed liquor.

The engineering and technology of the activated sludge system are reasonably well established, with systems implemented worldwide for the biological removal of C, N and/or P. Parallel to this development, significant advances have been made in the microbiological and biochemical areas of activated sludge. These advances have been driven by the development of new analytical techniques that allow microorganisms to be studied in situ in the activated sludge environment. However, there has been little cross-linking and overlap between the engineering and technology and microbiology and biochemistry paradigms. In particular, the information from the microbiology and biochemistry has not been integrated into the engineering and technology paradigm, to enable improved system design and optimization. One area that can form a starting point to build bridges between the two paradigm sets, is measurement of active biomass. The current design and simulation models invariably include active biomass for each organism group as fundamental parameters which define quantitatively the kinetic rates of the relevant biological processes. However, these parameters remain purely hypothetical because to date they have not been quantitatively measured; their acceptance is based on the consistency of model predicted results over a wide range of application. This paper describes developments in quantitative measurement of the heterotrophic and autotrophic active biomass concentrations within the engineering and technology paradigm, and the formulation of a multinational project which will attempt to link these measurements and the defined engineering environment to the new microbiological and biochemical analytical techniques. It is hoped that this project will facilitate integration of the two paradigms sets.

Bacteria↗

PubMed2004

Bioremediation process for sediments contaminated by heavy metals: feasibility study on a pilot scale.

The core stages of a sediment remediation process--the conditioning of dredged sludge by plants and the solid-bed leaching of heavy metals using microbially produced sulfuric acid--were tested on a pilot scale using a highly polluted river sediment. Conditioning was performed in 50 m3 basins at sludge depths of 1.8 m. During one vegetation period the anoxic sludge turned into a soil-like oxic material and became very permeable to water. Reed canary grass (Phalaris arundinacea) was found to be best suited for conditioning. Bioleaching was carried out in an aerated solid-bed reactor of 2000 L working volume using oxic soil-like sediment supplemented with 2% sulfur. When applying conditioned sediment, the oxidation of easily degradable organic matter by heterotrophic microbes increased the temperature up to 50 degrees C in the early leaching phase, which in turn temporarily inhibited the sulfur-oxidizing bacteria. Nevertheless, most of the metal contaminants were leached within 21 days. Zn, Cd, Mn, Co, and Ni were removed by 61-81%, Cu was reduced by 21%, while Cr and Pb were nearly immobile. A cost-effectiveness assessment of the remediation process indicates it to be a suitable treatment for restoring polluted sediments for beneficial use.

Biodegradation, Environmental↗

PubMed1998

A novel, non-redox-regulated NAD-dependent malate dehydrogenase from chloroplasts of Arabidopsis thaliana L.

We report a novel plastidic NAD-dependent malate dehydrogenase (EC 1. 1.1.37), which is not redox-regulated in contrast to its NADP-specific counterpart (EC 1.1.1.82). Analysis of isoenzyme patterns revealed a single NAD-MDH associated with highly purified chloroplasts isolated from Arabidopsis and spinach. A cDNA clone encoding the novel enzyme was found in the Arabidopsis EST data base by sorting all putative clones for NAD-dependent malate dehydrogenase. A derived amino acid sequence is very similar to mitochondrial and peroxisomal NAD-MDHs within the region coding for the mature protein but possesses a 80-amino acid long N-terminal domain with typical characteristics of a chloroplast transit peptide. In vitro synthesized labeled precursor protein was imported into the stroma of spinach chloroplasts and processed to a mature enzyme subunit of 34 kDa. Expressed in Escherichia coli, the recombinant enzyme exhibited the same distinctive isoelectric point of 5.35 as the original enzyme from Arabidopsis chloroplasts. Northern analysis revealed that the protein is expressed in both autotrophic and heterotrophic tissues. The findings reported here indicate that the "malate valve" operates not only in the illuminated chloroplasts but also in dark chloroplasts and in heterotrophic plastids and is therefore a general mechanism to maintain the optimal ratio between ATP and reducing equivalents in plastids.

Amino Acid Sequence↗

PubMed2003

One-carbon metabolism in plants. Regulation of tetrahydrofolate synthesis during germination and seedling development.

Tetrahydrofolate (THF) is a central cofactor for one-carbon transfer reactions in all living organisms. In this study, we analyzed the expression of dihydropterin pyrophosphokinase-dihydropteroate synthase (HPPK-DHPS) in pea (Pisum sativum) organs during development, and so the capacity to synthesize dihydropteroate, an intermediate in the de novo THF biosynthetic pathway. During seedling development, all of the examined organs/tissues contain THF coenzymes, collectively termed folate, and express the HPPK-DHPS enzyme. This suggests that each organ/tissue is autonomous for the synthesis of THF. During germination, folate accumulates in cotyledons and embryos, but high amounts of HPPK-DHPS are only observed in embryos. During organ differentiation, folate is synthesized preferentially in highly dividing tissues and in photosynthetic leaves. This is associated with high levels of the HPPK-DHPS mRNA and protein, and a pool of folate 3- to 5-fold higher than in the rest of the plant. In germinating embryos and in meristematic tissues, the high capacity to synthesize and accumulate folate correlates with the general resumption of cell metabolism and the high requirement for nucleotide synthesis, major cellular processes involving folate coenzymes. The particular status of folate synthesis in leaves is related to light. Thus, when illuminated, etiolated leaves gradually accumulate the HPPK-DHPS enzyme and folate. This suggests that folate synthesis plays an important role in the transition from heterotrophic to photoautotrophic growth. Analysis of the intracellular distribution of folate in green and etiolated leaves indicates that the coenzymes accumulate mainly in the cytosol, where they can supply the high demand for methyl groups.

Carbon↗

PubMed1999

Microbiological and geochemical characterization of fluvially deposited sulfidic mine tailings

The fluvial deposition of mine tailings generated from historic mining operations near Butte, Montana, has resulted in substantial surface and shallow groundwater contamination along Silver Bow Creek. Biogeochemical processes in the sediment and underlying hyporheic zone were studied in an attempt to characterize interactions consequential to heavy-metal contamination of shallow groundwater. Sediment cores were extracted and fractionated based on sediment stratification. Subsamples of each fraction were assayed for culturable heterotrophic microbiota, specific microbial guilds involved in metal redox transformations, and both aqueous- and solid-phase geochemistry. Populations of cultivable Fe(III)-reducing bacteria were most prominent in the anoxic, circumneutral pH regions associated with a ferricrete layer or in an oxic zone high in organic carbon and soluble iron. Sulfur- and iron-oxidizing bacteria were distributed in discrete zones throughout the tailings and were often recovered from sections at and below the anoxic groundwater interface. Sulfate-reducing bacteria were also widely distributed in the cores and often occurred in zones overlapping iron and sulfur oxidizers. Sulfate-reducing bacteria were consistently recovered from oxic zones that contained high concentrations of metals in the oxidizable fraction. Altogether, these results suggest a highly varied and complex microbial ecology within a very heterogeneous geochemical environment. Such physical and biological heterogeneity has often been overlooked when remediation strategies for metal contaminated environments are formulated.

Journal Article↗

PubMed1997

Bacterial communities of the rhizosphere and endorhiza associated with field-grown cucumber plants inoculated with a plant growth-promoting rhizobacterium or its genetically modified derivative.

The future use of genetically modified microorganisms in the environment will be dependent on the ability to asses potential or theoretical risks associated with their introduction into natural ecosystems. To assess potential risks, several ecological parameters must be examined, including the impact of the introduced genetically modified organism on the microbial communities associated with the environment into which the introduction will occur. A 2-year field study was established to examine whether the indigenous bacterial communities of the rhizosphere and endorhiza (internal root tissues) were affected differently by the introduction of an unaltered wild type and its genetically modified derivative. Treatments consisted of the wild-type strain Pseudomonas fluorescens 89B-27 and a bioluminescent derivative GEM-8 (89B-27::Tn4431). Cucumber root or seed samples were taken 0, 7, 14, 21, 35, and 70 days after planting (DAP) in 1994 and 0, 7, 14, 28, 42, and 70 DAP in 1995. Samples were processed to examine the bacterial communities of both the rhizosphere ad endorhiza. Over 7200 bacterial colonies were isolated from the rhizosphere Community structure at the genus level was assessed using genera richness and Hill's diversity numbers, N1 and N2. The aerobic-heterotrophic bacterial community structure at the genus level did not significantly vary between treatments but did differ temporally. The data indicate that the introduction of the genetically modified derivative of 89B-27 did not pose a greater environmental risk than its unaltered wild type with respect to aerobic-heterotrophic bacterial community structure.

Cucumis sativus↗

PubMed1998

Chlorophyll a availability affects psbA translation and D1 precursor processing in vivo in Synechocystis sp. PCC 6803.

Transcript accumulation and translation of psbA as well as processing of the D1 precursor protein were investigated in relation to chlorophyll availability in vivo in cyanobacterial strains lacking photosystem I (PS I). The psbA transcript level was almost independent of chlorophyll availability and was approximately 3-fold lower in darkness than in continuous light (5 microE m-2 s-1). Upon illumination, it reached a steady-state level within several hours. Upon growth under light-activated heterotrophic growth conditions (LAHG) in the PS I-less strain, D1 synthesis occurred immediately upon illumination. However, in PS I-less/chlL- cells, which lacked the light-independent chlorophyll biosynthesis pathway and had very low chlorophyll levels after LAHG growth, very little D1 synthesis occurred upon illumination, and the synthesis rate increased with time. This result suggests a translational control of D1 biosynthesis related to chlorophyll availability. Upon illumination, initially a high level of the nonprocessed D1 precursor was observed by pulse labeling and immunodetection in LAHG-grown PS I-less/chlL- cells but not in PS I-less cells. A significant amount of the D1 precursor eventually was processed to mature D1, and the half-life of the D1 precursor decreased as the chlorophyll content of the cells increased. The D1 processing enzyme CtpA was found to be present at similar levels regardless illumination or chlorophyll levels. We conclude that, directly or indirectly, chlorophyll availability is needed for D1 translation as well as for efficient processing of the D1 precursor.

Blotting, Western↗

PubMed1985

Vitamins: an evolutionary perspective.

Access to vitamins and the genetic endownment to utilize them maintain vitamin-dependent metabolic homeostasis in heterotrophs. Whereas the extent of adverse nutritional experiences has declined in modern human societies, phenocopies of deficiency diseases persist; accordingly, they have high heritability. The "vitamin-responsive hereditary metabolic diseases" identify DNA that specifies conserved apoenzyme domains interacting with coenzyme and the cellular processes providing access to coenzymes. Could heterozygosity at those loci also be a determinant of disease (or health) under certain circumstances?

Adaptation, Biological↗

PubMed2005

Enzymatic activity on sandy beaches of the Ligurian Sea (NW Mediterranean).

Enzymatic activity was measured on two beaches of the Ligurian Sea (NW Mediterranean) during late spring and summer 2003. The detected activities (leucine aminopeptidase, beta-glucosidase, alpha-glucosidase, and beta-N-acetylglucosaminidase) were related to the available organic substrates (proteins and carbohydrates) and to the bacterial community (expressed in terms of abundance, biomass, and frequency of cell division). The very low chlorophyll a concentrations (never higher than 40 ng g(-1)) suggested that heterotrophic microorganisms play a major role in the beach ecosystem. Enzymatic activities devoted to organic matter degradation were lower in the emerged part of the beaches and higher in the sites covered, permanently or temporarily, by seawater, suggesting that sea action enlivens the degradation processes. Leucine aminopeptidase ranged from 0.26 to 13.02 nmol g(-1)h(-1), and beta-glucosidase (the most expressed glycolytic enzyme) from 0.03 to 4.51 nmol g(-1)h(-1). Strong changes in the proteolytic/glycolytic activity ratio were observed, with a sudden rise in glycolysis during summer, leading to ratio values from about 30 down to 1. Thus, beaches were identified as preferential degradation sites, where very refractory compounds such as cellulose may also be efficiently processed.

Acetylglucosaminidase↗