Bio-Sensing of Cadmium(II) Ions Using Staphylococcus aureus†

Cadmium, as a hazardous pollutant commonly present in the living environment, represents an important risk to human health due to its undesirable effects (oxidative stress, changes in activities of many enzymes, interactions with biomolecules including DNA and RNA) and consequent potential risk, making its detection very important. New and unique technological and biotechnological approaches for solving this problems are intensely sought. In this study, we used the commonly occurring potential pathogenic microorganism Staphylococcus aureus for the determination of markers which could be used for sensing of cadmium(II) ions. We were focused on monitoring the effects of different cadmium(II) ion concentrations (0, 1.25, 2.5, 5, 10, 15, 25 and 50 μg mL−1) on the growth and energetic metabolism of Staphylococcus aureus. Highly significant changes have been detected in the metabolism of thiol compounds—specifically the protein metallothionein (0.79–26.82 mmol/mg of protein), the enzyme glutathione S-transferase (190–5,827 μmol/min/mg of protein), and sulfhydryl groups (9.6–274.3 μmol cysteine/mg of protein). The ratio of reduced and oxidized glutathione indicated marked oxidative stress. In addition, dramatic changes in urease activity, which is connected with resistance of bacteria, were determined. Further, the effects of cadmium(II) ions on the metabolic pathways of arginine, β-glucosidase, phosphatase, N-acetyl β-d-glucosamine, sucrose, trehalose, mannitol, maltose, lactose, fructose and total proteins were demonstrated. A metabolomic profile of Staphylococcus aureus under cadmium(II) ion treatment conditions was completed seeking data about the possibility of cadmium(II) ion accumulation in cells. The results demonstrate potential in the application of microorganisms as modern biosensor systems based on biological components

Bio-Sensing of Cadmium(II) Ions Using Staphylococcus aureus†

Cadmium, as a hazardous pollutant commonly present in the living environment, represents an important risk to human health due to its undesirable effects (oxidative stress, changes in activities of many enzymes, interactions with biomolecules including DNA and RNA) and consequent potential risk, making its detection very important. New and unique technological and biotechnological approaches for solving this problems are intensely sought. In this study, we used the commonly occurring potential pathogenic microorganism Staphylococcus aureus for the determination of markers which could be used for sensing of cadmium(II) ions. We were focused on monitoring the effects of different cadmium(II) ion concentrations (0, 1.25, 2.5, 5, 10, 15, 25 and 50 μg mL−1) on the growth and energetic metabolism of Staphylococcus aureus. Highly significant changes have been detected in the metabolism of thiol compounds—specifically the protein metallothionein (0.79–26.82 mmol/mg of protein), the enzyme glutathione S-transferase (190–5,827 μmol/min/mg of protein), and sulfhydryl groups (9.6–274.3 μmol cysteine/mg of protein). The ratio of reduced and oxidized glutathione indicated marked oxidative stress. In addition, dramatic changes in urease activity, which is connected with resistance of bacteria, were determined. Further, the effects of cadmium(II) ions on the metabolic pathways of arginine, β-glucosidase, phosphatase, N-acetyl β-d-glucosamine, sucrose, trehalose, mannitol, maltose, lactose, fructose and total proteins were demonstrated. A metabolomic profile of Staphylococcus aureus under cadmium(II) ion treatment conditions was completed seeking data about the possibility of cadmium(II) ion accumulation in cells. The results demonstrate potential in the application of microorganisms as modern biosensor systems based on biological components

Inorganic Cadmium Detection Using a Fluorescent Whole-Cell Bacterial Bioreporter

Cadmium pollution has become a serious environmental issue due to its toxicity and frequent entrance into environment components such as soil, water, and air via many anthropogenic sources. Over the last two decades, whole-cell bacterial bioreporters have been acknowledged as bio-sentinels for the determination of toxic heavy metals. Herein a sensitive and quite specific bacterial bioreporter was developed to cope with the need for the rapid and simple determination of cadmium. The construction and characterization of a fluorescence-based whole-cell cadmium bioreporter strain, Escherichia coli MG1655 (pBR-PzntA), was described which is based on the expression of green fluorescent protein under the control of the zntA gene promoter of heavy metal resistance determinant. The developed bioreporter was able determine cadmium at 5 mu g/L after 3.5 hours of induction in a defined medium while the cadmium detection limit was improved to 2 mu g/L after 1.5 hours by the use of an inorganic phosphate-limiting defined medium. Drastic changes in cadmium sensitivity were obtained between bioreporter cells induced at different growth phases. The maximum fluorescence performance was obtained for early exponential growth phase cells. This cadmium bioreporter was found to be more sensitive and specific to cadmium ions than to a wide range of heavy metals and was sensitive to only cadmium at drinking water quality standard concentrations. These findings will lead to future studies including integration of the bioreporter cells into a portable device to assess bioavailable cadmium levels in environmental samples which will provide a rapid and practical field detection technique

Luminescent bacterial sensor for cadmium and lead

A sensor plasmid was constructed by inserting the regulation unit from the cadA determinant of plasmid pI258 to control the expression of firefly luciferase. The resulting sensor plasmid pTOO24 is capable of replicating in Gram-positive and Gram-negative bacteria. The expression of the reporter gene as a function of added extracellular heavy metals was studied in Staphylococcus aureus strain RN4220 and Bacillus subtilis strain BR151. Strain RN4220(pTOO24) mainly responded to cadmium, lead and antimony, the lowest detectable concentrations being 10 nM, 33 nM and 1 nM respectively. Strain BR151(pTOO24) responded to cadmium, antimony, zinc and tin at concentrations starting from 3.3 nM, 33 nM, 1 mu M and 100 mu M, respectively. The luminescence ratios between induced and uninduced cells, the induction coefficients, of strains RN4220(pTOO24) and BR151(pTOO24) were 23-50 and about 5, respectively. These results were obtained with only 2-3 h incubation times. Freeze-drying of the sensor strains had only moderate effects on the performance with respect to sensitivity or induction coefficients. (C) 1998 Elsevier Science S.A. All rights reserved

Molecular modeling and characterization of Vibrio cholerae transcription regulator HlyU

BACKGROUND: The SmtB/ArsR family of prokaryotic metal-regulatory transcriptional repressors represses the expression of operons linked to stress-inducing concentrations of heavy metal ions, while derepression results from direct binding of metal ions by these 'metal-sensor' proteins. The HlyU protein from Vibrio cholerae is the positive regulator of haemolysin gene, it also plays important role in the regulation of expression of the virulence genes. Despite the understanding of biochemical properties, its structure and relationship to other protein families remain unknown. RESULTS: We find that HlyU exhibits structural features common to the SmtB/ArsR family of transcriptional repressors. Analysis of the modeled structure of HlyU reveals that it does not have the key metal-sensing residues which are unique to the SmtB/ArsR family of repressors, yet the tertiary structure is very similar to the family members. HlyU is the only member that has a positive control on transcription, while all the other members in the family are repressors. An evolutionary analysis with other SmtB/ArsR family members suggests that during evolution HlyU probably occurred by gene duplication and mutational events that led to the emergence of this protein from ancestral transcriptional repressor by the loss of the metal-binding sites. CONCLUSION: The study indicates that the same protein family can contain both the positive regulator of transcription and repressors – the exact function being controlled by the absence or the presence of metal-binding sites

Effect of environmental and nutritional conditions on the formation of single and mixed-species biofilms and their efficiency in cadmium removal

Remediation of contaminated water and wastewater using biosorption methods has attracted significant attention in recent decades due to its efficiency, convenience and minimised environmental effects. Bacterial biosorbents are normally deployed as a non-living powder or suspension. Little is known about the mechanisms or rates of bacterial attachment to surfaces and effect of various conditions on the biofilm development, as well as efficiency of living biofilms in the removal of heavy metals. In the present study, the effect of environmental and nutritional conditions such as pH, temperature, concentrations of phosphate, glucose, amino acid, nitrate, calcium and magnesium, on planktonic and biofilm growth of single and mixed bacterial cultures, were measured. Actinomyces meyeri, Bacillus cereus, Escherichia coli, Pseudomonas fluorescens strains were evaluated to determine the optimum biofilm growth conditions. The Cd(II) biosorption efficiencies of the mixed-species biofilm developed in the optimum growth condition, were investigated and modelled using Langmuir, Freundlich and Dubnin Radushkevich models. The biofilm quantification techniques revealed that the optimum concentration of phosphate, glucose, amino acid, nitrate, calcium and magnesium for the biofilm development were 25, 10, 1, 1.5, 5 and 0.5 g L−1, respectively. Further increases in the nutrient concentrations resulted in less biofilm growth. The optimum pH for the biofilm growth was 7 and alkaline or acidic conditions caused significant negative effects on the bacterial attachment and development. The optimum temperatures for the bacterial attachment to the surface were between 25 and 35 °C. The maximum Cd(II) biosorption efficiency (99%) and capacity (18.19 mg g−1) of the mixed-species biofilm, occurred on day 35 (Ci = 0.1 mg L−1) and 1 (Ci = 20 mg L−1) of biofilm growth, respectively. Modelling of the biosorption data revealed that Cd(II) removal by the living biofilm was a physical process by a monolayer of biofilm. The results of present study suggested that environmental and nutritional conditions had a significant effect on bacterial biofilm formation and its efficiency in Cd(II) removal

The Role of Bacillithiol in Gram-Positive Firmicutes

Significance: Since the discovery and structural characterization of bacillithiol (BSH), the biochemical functions of BSH-biosynthesis enzymes (BshA/B/C) and BSH-dependent detoxification enzymes (FosB, Bst, GlxA/B) have been explored in Bacillus and Staphylococcus species. It was shown that BSH plays an important role in detoxification of reactive oxygen and electrophilic species, alkylating agents, toxins, and antibiotics. Recent Advances: More recently, new functions of BSH were discovered in metal homeostasis (Zn buffering, Fe-sulfur cluster, and copper homeostasis) and virulence control in Staphylococcus aureus. Unexpectedly, strains of the S. aureus NCTC8325 lineage were identified as natural BSH-deficient mutants. Modern mass spectrometry-based approaches have revealed the global reach of protein S-bacillithiolation in Firmicutes as an important regulatory redox modification under hypochlorite stress. S-bacillithiolation of OhrR, MetE, and glyceraldehyde-3-phosphate dehydrogenase (Gap) functions, analogous to S-glutathionylation, as both a redox-regulatory device and in thiol protection under oxidative stress. Critical Issues: Although the functions of the bacilliredoxin (Brx) pathways in the reversal of S-bacillithiolations have been recently addressed, significantly more work is needed to establish the complete Brx reduction pathway, including the major enzyme(s), for reduction of oxidized BSH (BSSB) and the targets of Brx action in vivo. Future Directions: Despite the large number of identified S-bacillithiolated proteins, the physiological relevance of this redox modification was shown for only selected targets and should be a subject of future studies. In addition, many more BSH-dependent detoxification enzymes are evident from previous studies, although their roles and biochemical mechanisms require further study. This review of BSH research also pin-points these missing gaps for future research. Antioxid. Redox Signal. 28, 445–462

Biosensors for Environmental Monitoring

Real-time and reliable detection of molecular compounds and bacteria is essential in modern environmental monitoring. For rapid analyses, biosensing devices combining high selectivity of biomolecular recognition and sensitivity of modern signal-detection technologies offer a promising platform. Biosensors allow rapid on-site detection of pollutants and provide potential for better understanding of the environmental processes, including the fate and transport of contaminants.This book, including 12 chapters from 37 authors, introduces different biosensor-based technologies applied for environmental analyses

Comparative genomics of regulation of heavy metal resistance in Eubacteria

BACKGROUND: Heavy metal resistance (HMR) in Eubacteria is regulated by a variety of systems including transcription factors from the MerR family (COG0789). The HMR systems are characterized by the complex signal structure (strong palindrome within a 19 or 20 bp promoter spacer), and usually consist of transporter and regulator genes. Some HMR regulons also include detoxification systems. The number of sequenced bacterial genomes is constantly increasing and even though HMR resistance regulons of the COG0789 type usually consist of few genes per genome, the computational analysis may contribute to the understanding of the cellular systems of metal detoxification. RESULTS: We studied the mercury (MerR), copper (CueR and HmrR), cadmium (CadR), lead (PbrR), and zinc (ZntR) resistance systems and demonstrated that combining protein sequence analysis and analysis of DNA regulatory signals it was possible to distinguish metal-dependent members of COG0789, assign specificity towards particular metals to uncharacterized loci, and find new genes involved in the metal resistance, in particular, multicopper oxidase and copper chaperones, candidate cytochromes from the copper regulon, new cadmium transporters and, possibly, glutathione-S-transferases. CONCLUSION: Our data indicate that the specificity of the COG0789 systems can be determined combining phylogenetic analysis and identification of DNA regulatory sites. Taking into account signal structure, we can adequately identify genes that are activated using the DNA bending-unbending mechanism. In the case of regulon members that do not reside in single loci, analysis of potential regulatory sites could be crucial for the correct annotation and prediction of the specificity

Biosensorsüsteem mastiiti põhjustavate bakterite kiireks ja samaaegseks määramiseks piimas

Väitekirja elektrooniline versioon ei sisalda publikatsiooneMastiit on udarapõletik, mis enamasti tekib patogeensete mikroorganismide sattumisel läbi nisajuha udaraveerandisse, olles peamine lüpsilehmade nakkushaigus. Mastiidi poolt põhjustatud kahju kogu maailma 271 miljoni piimalehma kohta on hinnanguliselt 16-26 miljardit eurot aastas. Traditsiooniliste meetoditena mastiiti tekitavate bakterite identifitseerimiseks on tänapäeval kasutusel mikrobioloogilised analüüsid, mis võtavad aega 1-2 päeva ja laboratoorsetes tingimustes tehtavad patogeenide geenianalüüsid, mille tegemiseks kulub 6 tundi. Kuna ravi edukuse tagamiseks on selle täpsuse kõrval väga oluline ka selle võimalikult operatiivne alustamine, siis on vajalik välja töötada sellised analüüsimeetodid, mis võimaldavad patogeenide identifitseerimist oluliselt kiiremini kui praegu ning mida on võimalik kasutada farmides kohapeal. Tänasel päeval sellised meetodid puuduvad. Doktoritöö käigus töötati välja biosensorsüsteem ja mõõtemetoodika kolme peamise mastiiti tekitava patogeeni – Staphylococcus aureus’e Escherichia coli ja Streptococcus uberis’e määramiseks nii eraldi kui ühtlasi ka kõikide nimetatud patogeenide koos määramiseks. Biosensorsüsteemi konstrueerimisel uuriti selle tundlikkust, tööpiirkonda, selektiivsust ja sobivust rakendamiseks keerulistes maatriksites nagu piim. Biosensori selektiivsus kindla patogeeni määramiseks teiste bakterite olemasolul oli väga hea ning seda on võimalik kasutada mitme bakteri samaaegseks määramiseks. Väljapakutud biosensorsüsteemil põhinevat mõõtesüsteemi on võimalik kasutada loomade tervise automatiseeritud kontrolliks farmis kohapeal ja seeläbi kiiresti identifitseerida juba varajases staadiumis potentsiaalne haigus. Varajane haiguse avastamine aitab alustada koheselt kiiret ja sobivat ravi, parandades seeläbi looma heaolu ja piima kvaliteeti ning vähendada tootmiskulusid ja majanduslikku kahju.Mastitis, mostly caused by bacterial infection of the mammary gland, is a major health problem of dairy cows. The resulting decrease of milk production and reduction of its quality along with medication costs and probable premature culling of animals cause essential economic burden. The total mastitis caused losses in dairy industry are estimated to be 16–26 billion € annually in view of a global population of 271 million dairy cows. For the identification of mastitis-causing pathogens, the gold standard is microbiological culturing of bacteria, which in recent years has been partially replaced by polymerase chain reaction analyses of bacterial DNA. Although reliable, these methods require hours to obtain results, not allowing effective treatment of animals and optimal milk processing. Therefore to assure timely and correct treatment of animals, there is a great need for a method applicable for rapid automatic detection of mastitis causing pathogens in milk. A rapid method and an immuno-biosensing system have been proposed and applied for the rapid multiplex detection of major mastitis causing pathogens (Staphylococcus aureus, Escherichia coli and Streptococcus uberis) in milk. The key features of the biosensor development were its selectivity, sensitivity, working range and applicability for rapid automated analyses in complex biological sample matrix like milk. The selectivity of the biosensing system in the presence of several pathogens was very good and it was applicable for the multiplex detection of different pathogens. Based on the results of the present study, the proposed biosensing system has a great potential to serve as a tool for in-line automatic monitoring of milk and animal health in dairy farms