Overexpression of sgm 5’ UTR mRNA reduces gentamicin resistance in both Escherichia coli and Micromonospora melanosporea cells

16S rRNK metilaze su eksprimirane u većini bakterija koje proizvode antibiotike da bi se zaštitile od dejstva antibiotika putem metilacije 16S rRNK na pozicijama koje su bitne za njihovo dejstvo. Gen sgm koji je odgovoran za rezistenciju na sisomicin i gentamicin u soju Micromonospora zionensis, metilu je G1405 u okviru A mesta 16S rRNA gde se nalazi i CCGCCC heksanukleotid. Isti heksanukleotid se nalazi i 14 nukleotida ispred mesta vezivanja ribozoma na sgm informacionoj RNK. Predloženi model translacione regulacije sgm gena pretpostavlja da se Sgm protein vezuje za ovaj motiv kako na 16S rRNK, tako i na 5’ netranslirajućem regionu (UTR) sopstvene informacione RNK. 5’ UTR sekvenca je overeksprimirana na sgm informacionoj RNK sa skraćenim 3’ krajem i testiran je efekat na gentamicinsku rezistenciju u ćelijama E. coli i Micromonospora melanosporea. Overekspresija ove regulatorne sekvence dovodi do smanjenja rezistencije u oba testirana soja najverovatnije zbog titracije Sgm molekula od strane 5’ UTR-a.The 16S rRNA methylases are expressed by most of the antibiotic producing bacteria in order to protect themselves against antibiotics by methylation of 16S rRNA at positions which are crucial for their action. The sgm sisomicin-gentamicin resistance gene from Micromonospora zionensis methylates G1405 positioned in the A site of 16S rRNA, which includes a CCGCCC hexanucleotide. The same hexanucleotide is also present 14 nucleotides in front of the ribosome binding site of sgm mRNA. The model proposed for translational regulation of sgm assumes that Sgm binds to this motif, both on 16S rRNA and on the 5’ untranslated region (UTR) of its own mRNA. The 5’ UTR mRNA sequence was overexpressed on 3’-truncated sgm mRNA, and the effect on gentamicin resistance conferred by Sgm was tested in Escherichia coli and in Micromonospora melanosporea. Overexpression of the sgm mRNA regulatory region decreases the resistance to gentamicin in both E. coli and M. melanosporea. This effect is likely to be due to titration of Sgm molecules by the overexpressed 5’ UTR

Analysis of secondary structure within sgm and kgmB mRNA

Sgm metiltransferaza iz soja Micromonospora zionensis i KgmB metiltransferaza iz soja Streptoalloteichus tenebrarius ostvaruju rezistenciju na aminoglikozidne antibiotike metilacijom nukleotida na poziciji G1405 u okviru A mesta na 16S rRNK. Smatra se da je za autoregulaciju sgm gena odgovoran (C)CCGCCC motiv. Najverovatnije je ista sekvenca odgovorna i za autoregulaciju kgmB gena. Po kompjuterskoj predikciji, ovaj motiv, lociran u 5' netranslatirajućem regionu iRNK molekula oba gena, bi mogao učestvovati u formiranju sekundarne strukture tipa ukosnice. Kako Sgm i KgmB metiltransferaze jedna drugu autoregulišu, moguće je da prepoznaju iste cis elemente u iRNK molekulima. Eksperimenti ispitivanja strukture su, s jedne strane potvrdili prisustvo stabilne sekundarne strukture u okviru 5' netranslatirajućeg regiona iRNK molekula sgm gena, a sa druge, nisu dokazali postojanje modelovane sekundarne strukture u iRNK molekulu kgmB gena.Sgm methyltransferase from Micromonospora zionensis and KgmB methyltransferase from Streptoalloteichus tenebrarius are resistant to aminoglycoside antibiotics as a result of their ability to specifically methylate G1405 within the bacterial 16S rRNA A-site. The (C)CGCCC motif, assumed to be a regulatory sequence responsible for the autoregulation of the sgm gene, could most likely also be responsible for the autoregulation of the kgmB gene. This sequence, found within the 5' untranslated region of both sgm and kgmB mRNAs, as indicated by in silico prediction, may be involved in the formation of a specific stem-loop structure. Sgm and KgmB are mutually down-regulated and it is likely that they share the same cis-acting elements. Structure probing experiments confirmed the existence of a stable secondary structure within the 5' UTR of the sgm mRNA, while the analysis of kgmB mRNA failed to confirm the predicted structure

Analysis of secondary structure within sgm and kgmB mRNA

Sgm methyltransferase from Micromonospora zionensis and KgmB methyltransferase from Streptoalloteichus tenebrarius are resistant to aminoglycoside antibiotics as a result of their ability to specifically methylate G1405 within the bacterial 16S rRNA A-site. The (C)CGCCC motif, assumed to be a regulatory sequence responsible for the autoregulation of the sgm gene, could most likely also be responsible for the autoregulation of the kgmB gene. This sequence, found within the 5' untranslated region of both sgm and kgmB mRNAs, as indicated by in silico prediction, may be involved in the formation of a specific stem-loop structure. Sgm and KgmB are mutually down-regulated and it is likely that they share the same cis-acting elements. Structure probing experiments confirmed the existence of a stable secondary structure within the 5' UTR of the sgm mRNA, while the analysis of kgmB mRNA failed to confirm the predicted structure.

Photoactivatable Surface-Functionalized Diatom Microalgae for Colorectal Cancer Targeted Delivery and Enhanced Cytotoxicity of Anticancer Complexes

Systemic toxicity and severe side effects are commonly associated with anticancer chemotherapies. New strategies based on enhanced drug selectivity and targeted delivery to cancer cells while leaving healthy tissue undamaged can reduce the global patient burden. Herein, we report the design, synthesis and characterization of a bio-inspired hybrid multifunctional drug delivery system based on diatom microalgae. The microalgae's surface was chemically functionalized with hybrid vitamin B-12-photoactivatable molecules and the materials further loaded with highly active rhenium(I) tricarbonyl anticancer complexes. The constructs showed enhanced adherence to colorectal cancer (CRC) cells and slow release of the chemotherapeutic drugs. The overall toxicity of the hybrid multifunctional drug delivery system was further enhanced by photoactivation of the microalgae surface. Depending on the construct and anticancer drug, a 2-fold increase in the cytotoxic efficacy of the drug was observed upon light irradiation. The use of this targeted drug delivery strategy, together with selective spatial-temporal light activation, may lead to lower effective concentration of anticancer drugs, thereby reducing medication doses, possible side effects and overall burden for the patient

Bioleaching of copper from samples of old flotation tailings (Copper Mine Bor, Serbia)

Bioleaching of samples taken from depths of 10, 15, and 20 m from old flotation tailings of the Copper Mine Bor was conducted in shaken flasks using the extremely acidic water of Lake Robule as lixiviant. The yields of copper after five weeks of the bioleaching experiments were 68.34 +/- 1.21 % for the 15-m sample, 72.57 +/- 0.57 % for the 20-m sample and 97.78 +/- 5.50 % for the 10-m sample. The obtained results were compared to the results of acid leaching of the same samples and it was concluded that bioleaching was generally more efficient for the treatment of samples taken from depths of 10 and 20 m. The content of pyrite in the 20-in sample, which contained the highest amount of this mineral, was reduced after bioleaching. The benefits of this approach are recovery of substantial amounts of copper, reducing the environmental impact of flotation tailings and the application of abundant and free water from the acidic Robule Lake as lixiviant. The obtained results showed that bioleaching could be more efficient than acid leaching for copper extraction from flotation tailings with higher sulfide contents

In vitro antimicrobial activity and cytotoxicity of nickel(II) complexes with different diamine ligands

Three diamines, 1,3-propanediamine (1,3-pd), 2,2-dimethyl-1,3-propanediamine (2,2-diMe-1,3-pd) and (+/-)-1,3-pentanediamine (1,3-pnd), were used for the synthesis of nickel(II) complexes 1-3, respectively, of the general formula [Ni(L)(2)(H2O)(2)]Cl-2. The stoichiometries of the complexes were confirmed by elemental microanalysis, and their structures were elucidated by spectroscopic (UV-Vis and IR) and molar conductivity measurements. The complexes 1-3, along with NiCl2 center dot 6H(2)O and the diamine ligands, were evaluated against a panel of microbial strains that are associated with skin, wound, urinary tract and nosocomial infections. The obtained results revealed no significant activity of 1-3 against the investigated bacterial strains. On the other hand, they showed good antifungal activity against pathogenic Candida strains, with minimum inhibitory concentration (MIC) values in the range from 15.6 to 62.5 mu g mL(-1). The best anti-Candida activity was observed for complex 2 against C. parapsilosis, while the least susceptible to the effect of the complexes was C. krusei. The antiproliferative effect on normal human lung fibro-blast cell line MRC-5 was also evaluated in order to determine the therapeutic potential of nickel(II) complexes 1-3. These complexes showed lower negative effects on the viability of the MRC-5 cell line than the clinically used nystatin and comparable selectivity indexes to that of this antifungal drug

Polymerase chain reaction in the identification of periodontopathogens: A reliable and satisfactory method?

Aggregatibacter actinomycetemcomitans is considered one of the bacterial species of etiological importance in periodontitis. The aim of this study was to evaluate the serotype of A. actinomycetemcomitans in the subgingival biofilm in subjects with periodontal health and disease. Pooled samples of subgingival plaque were taken for culture-based identification of microorganisms. Colonies suspected to be A. actinomycetemcomitans were selected for molecular identification using either multiplex or conventional PCR in serotype-specific genotyping and 16S rRNA gene sequencing. In silico analysis showed that most selected colonies belong to the genus Campylobacter, although positive signals for serotypes of A. actinomycetemcomitans were obtained with these samples. Identification of A. actinomycetemcomitans by conventional PCR for 16S rRNA with one species-specific and one universal primer was inconclusive because an almost identical signal with Campylobacter gracilis was obtained. Although PCR-based methods for the identification of A. actinomycetemcomitans are more rapid, sequencing should not be omitted. [Projekat Ministarstva nauke Republike Srbije, br. 41008 and br. 173048

rRNA methyltransferases and their role in resistance to antibiotics

Metiltransferaze (MTaze), koje čine veliku proteinsku superfamiliju, kao donatora metil grupe najčešće koriste S-adenozil-L-metionin (SAM). SAM-zavisne MTaze metiluju nukleinske kiseline (DNK, RNK) i proteine, modulišući tako njihovu aktivnost, funkciju i strukturnu organizaciju. Metilacija G1405 ili A1408 baza u 16S rRNK mikroorganizama koji proizvode aminoglikozide obezbeđuje rezistenciju na sopstvene toksične proizvode. Ovaj mehanizam rezistencije je donedavno bio opisan samo kod proizvođača antibiotika. Od 2003. godine i kod patogenih bakterija beleži se neprestan porast rezistencije na aminoglikozide putem ovog mehanizma, što predstavlja veliku pretnju efikasnoj upotrebi aminoglikozida u kliničkoj praksi. Jedno od mogućih rešenja problema leži u razvoju novih jedinjenja koja bi efikasno delovala na nova mesta u okviru ribozoma. Drugi pristup rešavanju ovog problema uključuje razvoj inhibitora MTaza odgovornih za rezistenciju, sa idejom da se onemogući modifikacija bakterijske rRNK i na taj način vrati terapeutska efikasnost postojećim aminoglikozidima. Fundamentalna istraživanja vezana za proteinsku ekspresiju, potpuno razumevanje mehanizma rezistencije kao i razrešenje tercijarne strukture proteina su neophodan preduslov za primenu inhibitora 16S rRNK MTaza u medicini.Methyltransferases (MTases), a large protein superfamily, commonly use S-adenosyl-L-methionine (SAM) as the methyl group donor. SAM-dependant MTases methylate both nucleic acids (DNA, RNA) and proteins, and thus modulate their activity, function and folding. Methylation of G1405 or A1408 nucleotides of 16S rRNA in aminoglycoside-producing microorganisms confers the resistance to their own toxic product(s). This mechanism of resistance has been considered as unique to antibiotics producers until recently. Since 2003, methylation of 16S rRNA as a mechanism of resistance is increasingly emerging in pathogenic bacteria. This represents a major threat towards the usefulness of aminoglycosides in the clinical practice. A potential solution to the problem involves the design of novel compounds that would act against new ribosomal targets. The second approach to the issue includes the development of resistance MTases' inhibitors, with the idea to prevent them from modifying the bacterial rRNA, and thus reinstate the therapeutic power of existing aminoglycosides. As the latter approach has considerable potential, it is obvious that fundamental research related to protein expression, in-depth understanding of the mechanism of action and resolving a tertiary structure of 16S rRNAs MTases are prerequisites for application in medicine

Antimicrobial and anti-biofilm activity and biological decontamination efficiency of ED-1 emulsion

Antimicrobial and antibiofilm activity, as well as biological decontamination potential of emulsion ED-1, highly efficient in radiological decontamination of metal surfaces contaminated with uranium isotopes, was assessed. The antimicrobial potency of ED-1 was evaluated against 10 different microorganisms including four Gram-negative bacteria (Acinetobacter baumannii ATCC 19606, Escherichia coli NCTC 9001, Klebsiella pneumoniae ATCC 13803 and Pseudomonas aeruginosa NCTC 10662), four Gram-positive bacteria (Enterococcus faecalis ATCC 29212, Enterococcus faecium ATCC 6057, Listeria monocytogenes NCTC 11994, Staphylococcus aureus NCTC 6571) and two fungi (Candida albicans ATCC 10231 and Candida parapsilosis ATCC 22019). Although without strong bactericidal and fungicidal properties in standard agar diffusion assays, ED-1 effectively inhibited the growth of P. aeruginosa cells in liquid culture and more importantly, showed high potential to disperse P. aeruginosa biofilms. ED-1 was also capable to efficiently remove Bacillus subtilis ATCC 6633 spores in quantitative and a semi-quantitative biological decontamination tests on metal surfaces. Antimicrobial and antibiofilm activity and biological decontamination efficiency of ED-1 was comparable to and better than that of calcium hypochlorite solution or commercial decontaminant BX-24. This study highlighted the possibility to use ED-1, with up to 5-fold reduced amounts of calcium hypochlorite in comparison to currently used methodology, for both biological and radiological decontamination, resulting in both environmental and financial benefits

Polymerase chain reaction in the identification of periodontopathogens: A reliable and satisfactory method?

Aggregatibacter actinomycetemcomitans is considered one of the bacterial species of etiological importance in periodontitis. The aim of this study was to evaluate the serotype of A. actinomycetemcomitans in the subgingival biofilm in subjects with periodontal health and disease. Pooled samples of subgingival plaque were taken for culture-based identification of microorganisms. Colonies suspected to be A. actinomycetemcomitans were selected for molecular identification using either multiplex or conventional PCR in serotype-specific genotyping and 16S rRNA gene sequencing. In silico analysis showed that most selected colonies belong to the genus Campylobacter, although positive signals for serotypes of A. actinomycetemcomitans were obtained with these samples. Identification of A. actinomycetemcomitans by conventional PCR for 16S rRNA with one species-specific and one universal primer was inconclusive because an almost identical signal with Campylobacter gracilis was obtained. Although PCR-based methods for the identification of A. actinomycetemcomitans are more rapid, sequencing should not be omitted