Isopropyl-β-d-thiogalactopyranoside influences the metabolism of Escherichia coli

Summary Addition of isopropyl -D-thiogalactopyranoside (IPTG) to a strain of Escherichia coli with one lac promoter in its chromosome causes reduction in synthesis rate for a set of protiens. One of these proteins, designated H35, is a prominent cellular protein present only during exponential growth. Reduction of H35 synthesis is transient and delayed following an IPTG pulse. Cellular response to an IPTG pulse during exponential growth shares several features with a heat shock response. Significant increases in the specific growth rate of cells in both amino-acid-supplemented minimal medium and complex medium were observed for some IPTG concentrations relative to IPTG-free cultures. Other IPTG concentrations caused a reduction in specific growth rate.Offprint requests to: J. E. Bailey     

Cloning of the <Emphasis Type="Italic">cnr</Emphasis> operon into a strain of <Emphasis Type="Italic">Bacillaceae</Emphasis> bacterium for the development of a suitable biosorbent

In this study, a potential microbial biosorbent was engineered to improve its capacity to remediate heavy metal contaminated water resources. A Bacillaceae bacterium isolated from a mining area was transformed with a plasmid carrying the (pECD312)-based cnr operon that encodes nickel and cobalt resistance. The bioadsorption ability of the transformed strain was evaluated for removal of nickel from metallurgical water relative to the wildtype strain. Results showed that transformation improved the adsorption capacity of the bacterium by 37 % at nickel concentrations equivalent to 150 mg/L. Furthermore it was possible to apply prediction modelling to study the bioadsorption behaviour of the transformed strain. As such, this work may be extended to the design of a nickel bioremediation plant utilising the newly developed Bacillaceae bacterium as a biosorbent.     

Evidencing the role of lactose permease in IPTG uptake by Escherichia coli in fed-batch high cell density cultures

The lac-operon and its components have been studied for decades and it is widely used as one of the common systems for recombinant protein production in Escherichia coli. However, the role of the lactose permease, encoded by the lacY gene, when using the gratuitous inducer IPTG for the overexpression of heterologous proteins, is still a matter of discussion. A lactose permease deficient strain was successfully constructed. Growing profiles and acetate production were compared with its parent strain at shake flask scale. Our results show that the lac-permease deficient strain grows slower than the parent in defined medium at shake flask scale, probably due to a downregulation of the phosphotransferase system (PTS). The distributions of IPTG in the medium and inside the cells, as well as recombinant protein production were measured by HPLC-MS and compared in substrate limiting fed-batch fermentations at different inducer concentrations. For the mutant strain, IPTG concentration in the medium depletes slower, reaching at the end of the culture higher concentration values compared with the parent strain. Final intracellular and medium concentrations of IPTG were similar for the mutant strain, while higher intracellular concentrations than in medium were found for the parent strain. Comparison of the distribution profiles of IPTG of both strains in fed-batch fermentations showed that lac-permease is crucially involved in IPTG uptake. In the absence of the transporter, apparently IPTG only diffuses, while in the presence of lac-permease, the inducer accumulates in the cytoplasm at higher rates emphasizing the significant contribution of the permease-mediated transport.     

An “ex situ” microbial process for the removal of heavy metals from polluted soil: A case study of Ada rice field,Adani, Enugu State,Nigeria

An “ex situ” microbial method for the removal of heavy metals from soil is described. Elemental sulfur was added to generate the lixiviant in shaker flask experiments in which soil sampled from a polluted agricultural field was treated. The biotic oxidation of sulfur to sulfuric acid resulted in significant drop in pH of the bioleaching liquor from 6.94 to 1.8 after 50 days. In batches operated at very low (10 g/kg) sulfur concentrations, pH changed from 6.94 to 5.45. The 50 g/kg soil sulfur concentration was found to be most beneficial to the solubilization process because more than 95% of metals such as zinc (Zn), cadmium (Cd), and nickel (Ni) were recovered while approximately 67% of manganese (Mn) got solubilized. The least concentration of dissolved metals was lead (Pb) – (25%) and chromium (Cr) – (10%). Sulfate accumulation rose to 47% in samples spiked with 50 g/kg soil of sulfur. At lower sulfur concentrations, the sulfates generated were higher than the amount of sulfur added. The microbial process compared well to the abiotic process involving extraneous addition of sulfuric acid except that very high concentrations of acid had to be used. The treatment of the bioleaching wastewater promoted precipitation of the dissolved metals into insoluble hydroxides making discharge of the effluent into the environment safe. The leached soil recovered sufficiently for agricultural use after quick lime and animal manure was used to improve, stabilize, and restore its physical, chemical, and biological conditions.     

Nickel Bioremediation Potential of Bacillus thuringiensis KUNi1 and Some Environmental Factors in Nickel Removal

The present study endeavors to isolate a nickel (Ni)-resistant bacterial strain from an industrial waste–contaminated soil sample and to characterize the strain with a view to identify it and to assess its ability to remove Ni from the medium or detoxify it. The final objective is to use the strain as an agent to bioremediate Ni contamination. As an outcome, a Ni-resistant bacterial strain (KUNi1) had been isolated from such a soil that could tolerate a maximum of 7.5 and 10 mM Ni concentrations, depending on the type of medium used. The strain also showed multimetal resistance. It was found to be resistant to zinc (Zn), copper (Cu), cobalt (Co), and cadmium (Cd). However, the degree of resistance to the individual metal was variable, as determined by assessing the minimum inhibitory concentration (MIC) of each metal against the strain. The order of resistance was Ni > Zn = Cu = Co > Cd. The strain removed a significant percentage (82%) of Ni from the medium during in vitro culture, whereas dead cell mass had an insignificant role in Ni removal. The quantum of Ni removal by the strain was interfered with when the other metals (Zn, Cu, Co, and Cd) were present either singly with Ni or in combination with other metals. However, the degree of interference varied with individual metal. The factors that influenced the quantum of Ni removal were ambient pH, initial cell density, and presence of other toxic metals. The strain was identified as Bacillus thuringiensis on the basis of its biochemical characteristics and 16s rDNA sequence analysis.     

Proteomic analysis of the response of the plant growth-promoting bacterium Pseudomonas putida UW4 to nickel stress

Background  

Plant growth-promoting bacteria can alleviate the inhibitory effects of various heavy metals on plant growth, via decreasing levels of stress-induced ethylene. However, little has been done to detect any mechanisms specific for heavy metal resistance of this kind of bacteria. Here, we investigate the response of the wild-type plant growth-promoting bacterium Pseudomonas putida UW4 to nickel stress using proteomic approaches. The mutant strain P. putida UW4/AcdS^-, lacking a functional 1-aminocyclopropane-1-carboxylic acid deaminase gene, was also assessed for its response to nickel stress.     

Physiological effects of translation initiation factor IF3 and ribosomal protein L20 limitation inEscherichia coli

To investigate the physiological roles of translation initiation factor IF3 and ribosomal protein L20 inEscherichia coli, theinfC, rpmI andrpIT genes encoding IF3, L35 and L20, respectively, were placed under the control oflac promoter/operator sequences. Thus, their expression is dependent upon the amount of inducer isopropyl thiogalactoside (IPTG) in the medium. Lysogenic strains were constructed with recombinant lambda phages that express eitherrpmI andrplT orinfC andrpmI in trans, thereby allowing depletion of only IF3 or L20 at low IPTG concentrations. At low IPTG concentrations in the IF3-limited strain, the cellular concentration of IF3, but not L20, decreases and the growth rate slows. Furthermore, ribosomes run off polysomes, indicating that IF3 functions during the initiation phase of protein synthesis in vivo. During slow growth, the ratio of RNA to protein increases rather than decreases as occurs with control strains, indicating that IF3 limitation disrupts feedback inhibition of rRNA synthesis. As IF3 levels drop, expression from an AUU-infC-lacZ fusion increases, whereas expression decreases from an AUG-infC-lacZ fusion, thereby confirming the model of autogenous regulation ofinfC. The effects of L20 limitation are similar; cells grown in low concentrations of IPTG exhibited a decrease in the rate of growth, a decrease in cellular L20 concentration, no change in IF3 concentration, and a small increase in the ratio of RNA to protein. In addition, a decrease in 50S subunits and the appearance of an aberrant ribosome peak at approximately 41–43S is seen. Previous studies have shown that the L20 protein negatively controls its own gene expression. Reduction of the cellular concentration of L20 derepresses the expression of anrplT-lacZ gene fusion, thus confirming autogenous regulation by L20.     

Nickel requirement for chemolithotrophic growth in hydrogen-oxidizing bacteria

In a comparative study the requirement of several strains of autotrophic hydrogen-oxidizing bacteria for nickel was examined. Autotrophic growth was studied both in liquid media, previously freed from trace metals; and on solidified media, using a plate diffusion assay. The latter assay was based on the observation that EDTA causes complete inhibition of autotrophic growth on agar medium as a result of nickel deficiency. Nickel was shown to be required as a trace element in five strains of Alcaligenes eutrophus, in two strains of Xanthobacter autotrophicus, in Pseudomonas flava, in Arthrobacter spec. 11X and in strain 12X. In these bacteria nickel was not replaceable by cobalt, copper, manganese or zinc ions. No significant nickel requirement was detected by these methods, however, for Paracoccus denitrificans and Nocardia opaca 1b.     

Physiological and toxicological characterization of an engineered whole-cell biosensor

Bioluminescence-based bacterial biosensors are often reported as reliable and efficient tools for risk assessment and environmental monitoring. However, there are few data comparing the metabolism of genetically engineered strains to the corresponding wild type. A pollutant-degrading bacterium capable of mineralising 2,4-dichlorophenoxyacetic acid (2,4-D), Burkholderia sp. strain RASC c2, was genetically engineered to produce light constitutively and tested for assessing the main causes of biodegradation constraint affected by growth rates, toxicity, bioavailability and metal speciation in complex environments. This research focuses on such aspects by characterizing two pollutant-degrading isolates, the wild type and the genetically engineered biosensor (lux-marked). Degradation and growth rates of both isolates were assessed with different concentrations of 2,4-D as the sole carbon source. Kinetic rates were affected by initial concentration of substrate and isolates showed distinct growth rates at different 2,4-D concentrations. Toxic effects of zinc and copper were also comparatively assessed using a dehydrogenase assay and light output. The isolates were sensitive to both metals and at similar EC(50) values. Therefore, bioluminescence response of the lux-marked isolate accurately reflected the toxic response of the parental organism towards zinc and copper, making it an ideal test-organism for assessing toxicity in the context of pollutant mineralization.     

Plasmids in the bacterial assemblage of a dystrophic Lake: Evidence for plasmid-encoded nickel resistance

Sixty-two aerobic bacterial strains isolated from the unproductive dystrophic Lake Skärshultsjön (South Sweden) were screened for plasmids. The lake is considered to be an extreme environment because of its high concentration of persistent but nontoxic humic compounds. One-third of the isolates harbored multiple plasmids usually of similar high molecular weights (>25 Mdal). The plasmid-bearing strains were members of the common aquatic taxaPseudomonas spp.,Acinetobacter sp.,Alcaligenes sp.,Aeromonas/Vibrio group, andEnterobacteriaceae (taxonomy is tentative). The majority of isolates displayed multiple resistance to antibiotics and heavy metals. Some of them were capable of degrading aromatic compounds. Three isolates were chosen for curing experiments. Only strain S-68, anAlcaligenes sp., could be cured of one of its two plasmids. It harbored the two cryptic plasmids pQQ32 and pQQ70 of 32 and ca. 70 Mdal, and the latter was segregated during ethidium bromide treatment. Parental strain S-68 was capable of degrading some of nonchlorinated phenolic compounds and displayed resistance to a broad spectrum of antibiotics and the heavy metals Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, and Hg²⁺. Derivative strain S-68-41 lost its resistance to nickel, suggesting segregated plasmid PQQ70 coded for nickel resistance. Transformation experiments to restore nickel resistance in the cured derivative strain were not successful.     

Thiobacillus ferrooxidans response to copper and other heavy metals: growth, protein synthesis and protein phosphorylation

Respirometric experiments demonstrated that the oxygen uptake by Thiobacillus ferrooxidans strain LR was not inhibited in the presence of 200 mM copper. Copper-treated and untreated cells from this T. ferrooxidans strain were used in growth experiments in the presence of cadmium, copper, nickel and zinc. Growth in the presence of copper was improved by the copper-treated cells. However, no growth was observed for these cells, within 190 h of culture, when cadmium, nickel and zinc were added to the media. Changes in the total protein synthesis pattern were detected by two-dimensional polyacrylamide gel electrophoresis for T. ferrooxidans LR cells grown in the presence of different heavy metals. Specific proteins were induced by copper (16, 28 and 42 kDa) and cadmium (66 kDa), whereas proteins that had their synthesis repressed were observed for all the heavy metals tested. Protein induction was also observed in the cytosolic and membrane fractions from T. ferrooxidans LR cells grown in the presence of copper. The level of protein phosphorylation was increased in the presence of this metal.     

Mass balance on water column trace metals in a free-surface-flow-constructed wetlands in Sacramento, California

A mass balance has been performed on trace metals concentrations and hydrology observed between 1994 and 1996 at the Sacramento Demonstration Constructed Wetlands using a first-order areal plug flow model. Water losses to infiltration and evapotranspiration from a typical cell are estimated to average 35 and 7% of influent flow, respectively. The wetlands effluent metals concentrations consistently meet proposed discharge criteria. Annual total mass loadings for all trace metals average 14.0 kg ha⁻¹ yr⁻¹, 88% of which consists of zinc, copper, and nickel. Effluent metals leaving the wetland average 3.1 kg ha⁻¹ yr⁻¹, 79% of which consists of the same three metals. Annual vegetation harvest events do not appear to account for more than 5% of annual trace metals mass removal, although harvest does appear to represent a significant loss pathway for some metals like mercury, lead, nickel, and chromium. Metals mass removals resulting from first-order removal interactions within the wetland range from 27 to 81%, with the exception of arsenic and nickel which display poor mass removals in part due to their high dissolved concentrations. An average of 7.6 kg ha⁻¹ yr⁻¹, or 54% of influent metals loadings, is sequestered within the internal wetland compartments.     

Metal concentrations in water,sediment and sharptooth catfish Clarias gariepinus from three peri-urban rivers in the upper Manyame catchment,Zimbabwe

Concentrations of zinc, cadmium, chromium, nickel, lead, copper and iron were measured in flowing water, riverbed sediments and tissues of sharptooth catfish Clarias gariepinus from three rivers in the upper Manyame catchment over seven months in 2008–2009. The Manyame and Mukuvisi rivers are severely polluted by industrial and domestic effluent, whilst the Gwebi River is not influenced by urban effluent. Key water quality parameters, including dissolved oxygen and conductivity, clearly showed a pollution gradient in the Mukuvisi and Manyame rivers, but water quality in the Gwebi River was good. Levels of zinc, iron, copper, nickel and lead in fish tissues from the three rivers sampled were unusually high, with zinc and iron concentrations being the highest in all the tissues. This was also positively correlated with the concentrations of these metals in water and sediments. Notable differences existed between the water (zinc and copper) and sediments (iron and zinc) of each river. The relatively high metal concentrations in the Gwebi River, as well as conductivity and dissolved ions, were explained by the geological influence of the Great Dyke in its subcatchment. Metals are bound in the sediment but these can be rapidly mobilised into water if environmental changes occur, therefore efforts to monitor and prevent further water quality deterioration are required. The results of this study may have significant negative implications for aquatic organisms and for human health through fish consumption and therefore risk assessment investigations are imperative.     

Production of phloroglucinol by Escherichia coli using a stationary-phase promoter

Escherichia coli was metabolically engineered using a new host-vector system to produce phloroglucinol. The key biosynthetic gene phlD (encoding a type III polyketide synthase) from Pseudomonas fluorescens was expressed in E. coli using the stationary-phase promoter of the fic gene and a high-copy plasmid. In shake-flasks, the engineered strain produced phloroglucinol up to 0.28 g/l with a productivity of 0.014 g/l h. About 9.2% of the glucose consumed was converted to phloroglucinol after 20 h. Compared with the widely used inducible T7 promoter system, this strain did not require IPTG induction and the final titer of phloroglucinol was 22% higher.     

一株拮抗香蕉枯萎病的内生细菌的分离及其几丁质酶基因信号肽的分泌活性分析

目的：旨在分离并选择一株香蕉内生细菌作为内生基因工程生防菌,并克隆其几丁质酶基因的信号肽序列。方法：从香蕉植株杆下部分离并选择了一株拮抗香蕉枯萎病且具有分泌几丁质酶能力的内生细菌,对该菌株进行了形态观察、生理生化测定和16S rDNA序列分析,克隆了其几丁质酶基因的编码序列并预测了其信号肽,构建了含有信号肽和不含信号肽的几丁质酶的表达菌株BL-chi1和BL-chi2。结果：结合形态观察、生理生化特征和16S rDNA序列比对分析确定该菌株为Klebsiella属,将该菌株命名为KKWB 5;BL-chi1和BL-chi2经IPTG诱导后,均表达了与预期蛋白大小一致的蛋白,同时BL-chi1诱导后的培养基上清中出现一条约45kDa的条带,而BL-chi2和空载体的BL-pET22b诱导后的培养基上清中均无此条带;几丁质水解试验发现,BL-chi1诱导后的培养基上清中的蛋白经浓缩和纯化后都能在几丁质平板上形成透明水解圈。结论：该几丁质酶的信号肽能被BL21（DE3）所识别,将几丁质酶分泌到培养基中,并且分泌的几丁质酶具有水解几丁质的生物学活性。内生菌KKWB-5的分离及其几丁质酶分泌信号肽序列的克隆为进一步构建内生工程菌来防治香蕉枯萎病打下了基础。     

Toxic effects of lead and nickel nitrate on rat liver chromatin components

The biological activity of heavy metals is related to their physicochemical interaction with biological receptors. In the present study, the effect of low concentrations of nickel nitrate and lead nitrate (<0.3 mM) on rat liver soluble chromatin and histone proteins was examined. The results showed that addition of various concentrations of metals to chromatin solution preceded the chromatin into aggregation and precipitation in a dose-dependant manner; however, the extent of absorbance changes at 260 and 400 nm was different between two metals. Gel electrophoresis of histone proteins and DNA of the supernatants obtained from the metal-treated chromatin and the controls revealed higher affinity of lead nitrate to chromatin compared to nickel nitrate. Also, the binding affinity of lead nitrate to histone proteins free in solution was higher than nickel. On the basis of the results, it is concluded that lead reacts with chromatin components even at very low concentrations and induce chromatin aggregation through histone-DNA cross-links. Whereas, nickel nitrate is less effective on chromatin at low concentrations, suggesting higher toxicity of lead nitrate on chromatin compared to nickel.     

Nickel and cobalt resistance of various bacteria isolated from soil and highly polluted domestic and industrial wastes

Abstract From enrichment cultures in the presence of 1 mM NiCl₂ 200 strains of aerobic bacteria were isolated from 50 samples collected in the metal-processing industry, waste water treatment plants and from solid waste, highly polluted by heavy metals. The strains isolated were characterized with respect to their substrate spectrum and resistance to nickel, cobalt, zinc and cadmium salts and assigned to 21 groups. One representative of each group was described with respect to cell morphology. All strains were Gram-negative, non-sporing rods or cocci. The highest concentrations of nickel, cobalt, zinc, cadmium, copper, mercury, and silver allowing growth on solid media were estimated. Two strains were able to grow at 20 mM NiCl₂ and CoCl₂, one strain tolerated 12 mM and one 7.5 mM concentrations of these salts.
Fifteen out of 21 strains contained at least one plasmid two contained two plasmids. The plasmid sizes varied between 50 and 340 kbp, except strain 10A, which contained a miniplasmid (2.6 kbp). Attempts to cure four selected strains by exposure to mitomycin C or growth at elevated temperature failed.
By helper-assisted and unassisted conjugation the plasmids of strain 31A were shown to carry nickel and cobalt resistance determinants. Alcaligenes eutrophus strains H16 and N9A and denative of strain CH34 lacking one or both of its native metal resistance plasmids were used as recipients. Both plasmids, p TOM8 and pTOM9, of strain 31A carried resistance properties which were expressed in all recipients except. A. eutrophus H16, in which only nickel resistance was expressed.
Plasmid pTOM3 residing in strain 10A could not be transferred as such. However, transconjugants derived from helper (pULB113)-assisted matings carried co-integrates of various sizes and were resistant to nickel and cobalt.     

Function of a Novel Cadmium-Induced YodA Protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Cells of Escherichia coli increase greatly the synthesis of a small primarily cytoplasmic protein as soon as the cell growth rate falls below the maximal growth rate supported by cadmium exposure, after which the mature product is exported to the periplasm. This protein was previously identified as the product of the E. coli yodA open reading frame. We now report the isolation of an E. coli mutant defective in YodA synthesis because of insertional inactivation of the corresponding gene. In experiments to test the ability of both the wild-type and yodA mutant E. coli cells to bind cadmium, we have used γ-labeled [¹⁰⁹Cd]. Whereas the wild-type E. coli strain was able to bind metal, the yodA mutant strain failed to do so. In addition, analysis of such a mutant demonstrated that it grows at a rate distinguishable from that of the isogenic parent in the presence of cadmium ions. However, challenging cells with hydrogen peroxide and additional metals such as zinc, copper, cobalt, and nickel did not significantly affect the growth rate of the mutant. This growth phenotype was found to be the result of the loss of its ability to bind cadmium. These results suggest that the role of YodA protein might be to decrease the concentration level of cadmium ions in E. coli cells during cadmium stress by its ability to bind heavy metal.