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The self-aggregation and thermodynamic properties of three cationic surfactants as quaternary ammonium salts have been investigated. The physicochemical properties of compounds containing quinuclidin-3-ol with even-number of carbon atoms (10, 12 and 14) in the hydrophobic tail were measured by conductivity, dynamic light scattering (DLS) and Zeta-potential measurements. DLS and Zeta- potential measurements show similar size distribution for all surfactants with excellent uniformity, and Zeta-potential increases significantly with increase in the size of hydrocarbon tail. The critical micellar concentration (c.m.c.) and the degree of micelle ionization () were determined using conductivity measurements. The c.m.c. values of surfactants were found to be between 3.4 to 23.8.10-3 M. The standard Gibbs free energy (∆G_mic^o) was derived from conductivity measurements and suggests that surfactants containing longer chains spontaneously form micelles. The antioxidative properties of these cationic surfactants were evaluated using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC) assays. Among tested samples, N-tetradecyl-3- hydroxyquinuclidinium bromide (QOH-C14) exhibited the highest antioxidative potential (388.30 nmol (TE) equivalents mL-1), which was further investigated by the DNA nicking assay.

Quaternary ammonium compounds (QACs) are amphiphilic molecules displaying a broad-spectrum of antibacterial activity. QACs are commonly used antiseptics in industrial, home and hospital settings. Given the emergence of the QAC-resistant bacteria, there is an urgent need to design new QACs with good antimicrobial activity, able to escape the host resistance mechanism. Therefore, a series of QACs derived from quinuclidine-3-ol and an alkyl chain of variable length (QOH-C3 to -C14), was designed and synthesized. The antimicrobial potential of the new monoquaternary QACs was surveyed against seventeen strains of emerging food spoilage and pathogenic microorganisms, including clinical multidrug-resistant ESKAPE isolates. The QOH-C14 proved to have the strongest antimicrobial activity. It was highly active against all pathogens tested, particularly against the Gram-positive bacteria with minimal inhibitory concentrations (MICs) ranging from 0.06 to 3.9 μg/mL, and fungi exerting the MIC90 between 0.12 and 3.9 μg/mL. The potency of QOH-C14, confirmed that alkyl chains are an important part of the structure with their lengths playing a critical role in bioactivity of these compounds. The atomic force microscopy images show the disruption of a cell membrane upon the treatment with QOH-C14. These results were additionally confirmed by flow cytometry and fluorescence microscopy. A relatively low toxicity toward healthy human cells underline that QOH-C14 has a potential as new QAC antimicrobial candidate.

The chemical profile, antiproliferative, antioxidant and antiphytoviral activities of the species Hypericum perforatum ssp. veronense (Schrank) H. Lindb. (Clusiaceae) were investigated. Free volatiles were isolated and the chemical composition was determined in the lipophilic fraction (essential oil) and for the first time in the water fraction (hydrosol). The aim is to provide phytochemical data for H. perforatum ssp. veronense useful for distinguishing ssp. veronense from ssp. angustifolium, as there are taxonomic disagreements between them and the composition of the secretory products may be helpful in this respect. In the essential oil, the most abundant compounds identified were α-pinene and n-nonane, while in the hydrosol, myrtenol, carvacrol and α-pinene were the most abundant. Overall, the class of monoterpenes and oxygenated monoterpenes dominated in the EO and hydrosol samples. The essential oil showed high antioxidant activity, in contrast to the antiproliferative activity, where the hydrosol showed exceptional activity against three cancer cell lines: Hela (cervical cancer cell line), HCT116 (human colon cancer cell line) and U2OS (human osteosarcoma cell line). Both the essential oil and hydrosol showed antiphytoviral activity against tobacco mosaic virus infection on the local host plants. This is the first report dealing with biological activities of hydrosol of H. perforatum ssp. veronense, and the obtained results suggest that this traditional medicinal plant is a valuable source of volatiles with promising antiproliferative, antioxidant and antiphytoviral activities.

Mitophagy is a conserved intracellular catabolic process responsible for the selective removal of dysfunctional or superfluous mitochondria to maintain mitochondrial quality and need in cells. Here, we examine the mechanisms of receptor-mediated mitophagy activation, with the focus on BNIP3L/NIX mitophagy receptor, proven to be indispensable for selective removal of mitochondria during the terminal differentiation of reticulocytes. The molecular mechanisms of selecting damaged mitochondria from healthy ones are still very obscure. We investigated BNIP3L dimerization as a potentially novel molecular mechanism underlying BNIP3L-dependent mitophagy. Forming stable homodimers, BNIP3L recruits autophagosomes more robustly than its monomeric form. Amino acid substitutions of key transmembrane residues of BNIP3L, BNIP3L(G204A) or BNIP3L(G208V), led to the abolishment of dimer formation, resulting in the lower LC3A-BNIP3L recognition and subsequently lower mitophagy induction. Moreover, we identified the serine 212 as the main amino acid residue at the C-terminal of BNIP3L, which extends to the intermembrane space, responsible for dimerization. In accordance, the phosphomimetic mutation BNIP3L(S212E) leads to a complete loss of BNIP3L dimerization. Thus, the interplay between BNIP3L phosphorylation and dimerization indicates that the combined mechanism of LIR phosphorylation and receptor dimerization is needed for proper BNIP3L-dependent mitophagy initiation and progression. Abbreviations: AMBRA1: autophagy and beclin 1 regulator 1; Baf A1: bafilomycin A(1); BH3: BCL2 homology 3; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CoCl(2): cobalt (II) chloride; FKBP8: FKBP prolyl isomerase 8; FUNDC1: FUN14 domain containing 1; GABARAP: GABA type A receptor-associated protein; GST: glutathione S-transferase; IMM: inner mitochondrial membrane; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OMM: outer mitochondrial membrane; PHB2: prohibitin 2; PI: propidium iodide; PINK1: PTEN induced kinase 1; TM: transmembrane domain; TOMM20: translocase of outer mitochondrial membrane 20

This study is aimed to better understand the bactericidal mode of action of silver nanoparticles. Here we present the production and characterization of laser-synthesized silver nanoparticles along with growth curves of bacteria treated at sub-minimal and minimal inhibitory concentrations, obtained by optical density measurements. The main effect of the treatment is the increase of the bacterial apparent lag time, which is very well described by the novel growth model as well as the entire growth curves for different concentrations. The main assumption of the model is that the treated bacteria uptake the nanoparticles and inactivate, which results in the decrease of both the nanoparticles and the bacteria concentrations. The lag assumes infinitive value for the minimal inhibitory concentration treatment. This apparent lag phase is not postponed bacterial growth. It is a dynamic state in which the bacterial growth and death rates are close in value. Our results strongly suggest that the predominant mode of antibacterial action of silver nanoparticles is the penetration inside the membrane.

Species that belong to the genus Allium have been widely used for human food and traditional medicine. Their beneficial health effects, as well as the specific aroma, are associated with their bioactive chemical compounds, such as sulfur compounds and flavonoids. Gas chromatography and mass spectrometry (GC–MS) and reverse-phase high-performance liquid chromatography (reverse-phase HPLC) were used to identify organosulfur and amino acid content of triploid hybrid onion, Allium cornutum Clement ex Visiani, 1842, and common onion, Allium cepa L. Allium extracts were tested for their antiproliferative activity in three human cancer cell lines (HeLa, HCT116, and U2OS). DNA fragmentation and DAPI staining analysis were performed on HeLa cells to evaluate the effect of extracts on DNA damage and cell morphology. The mRNA expression of p53, Bax, and Caspase-3 genes involved in apoptosis were analyzed by real-time PCR. Using GC–MS, 27 compounds were found in two Allium species headspaces. Differences were noted among the main compound abundance in the headspace (although the major thiols and disulfides were qualitatively identic in both Allium species) and dipropyl disulfide, diisopropyl trisulfide, and (Z)-prop-1-enyl propyl trisulfide were predominant sulfides. Identification of amino acids and their quantities were determined by reverse-phase HPLC. Most abundant amino acids in both onions were arginine (Arg) and glutamic acid (Glu). The results of cytotoxicity testing confirmed antiproliferative effects of both species. The DNA fragmentation assay, DAPI staining and real time PCR analysis confirmed that A. cornutum and A. cepa extracts induced apoptosis in HeLa cells. This study presents the evidence for possible therapeutic use of A. cornutum and A. cepa extracts against human cervical carcinoma cell line.

The Atlantic bluefin tuna (ABFT; Thunnus thynnus) today represents one of the economically most important species for Croatian fisheries industry. Although the most diverse and abundant parasitofauna is usually found in the largest specimens of wild ABFT, the opposite was observed in captivity where parasite populations significantly decline by the end of the farming cycle. Copepod Brachiella thynni, is a skin parasite frequently parasitizing tuna, whose population also decreases in number throughout the rearing process. In order to better understand the immunity mechanisms underlying ABFT reaction to B. thynni infection, we studied expression profiles of immunity related genes; interleukin 1β (il1β), tumour necrosis factors (tnfα1, tnfα2), complement component 4 (c4) and caspase 3 (casp3), in peripheral blood leukocytes (PBLs) during in vitro stimulation by B. thynni protein extracts (i.e. antigens) and in infected tissues at B. thynni parasitation site. Finally, a histopathological analysis of semi-thin and ultra-thin sections of tissues surrounding B. thynii attachment site was performed to evaluate the severity of parasite-induced lesions and identify involved cell lineages. In vitro stimulation of ABFT PBLs with B. thynii antigens caused a dose-depended upregulation of selected genes, among which tnfα1 showed the highest induction by both concentrations of B. thynni protein extract. However, targeted genes were not significantly upregulated in the infected tissue. Also, no significant alterations in ultrastructure of epithelial layers surrounding B. thynii attachment site were noticed, except local tissue erosion, necrosis of squamous epithelium and proliferation of rodlet and goblet cells. Our results suggest that B. thynii has evolved strategies to successfully bypass both innate immune response and the connective-tissue proliferation processes. Therefore, the observed disappearance of this copepod by the end of the rearing process is more likely related to its limited lifespan on the host and its inability to complete the life cycle in the rearing cages, rather than host's reaction.

We present a simple growth model which was developed to explain Escherichia coli growth in batch culture. Optical density measurements are used to obtain E. coli growth curves for different inoculum sizes and nutrients concentrations. The model is described by two nonlinear mutually dependent differential equations that describe time evolution of bacteria and nutrients concentration. Introduction of the negative bacterium– bacterium interaction term is specific for the model and leads to the population decay. The proposed model describes entire experimental growth curves. The growth rate, as a function of initial nutrients concentration, follows the Monod function, whilst during the growth it decreases proportionally with the concentration of nutrients. The parameters in our equations can be related to the parameters of the logistic model. The proposed model can be applied to different E. coli strains and, because of the universality of the equations, might be applied to other bacterial strains.

Quaternary ammonium compounds (QACs) are antimicrobial agents displaying a broad spectrum of activity due to their mechanism of action targeting the bacterial membrane. The emergence of bacterial resistance to QACs, especially in times of pandemics, requires the continuous search for new and potent QACs structures. Here we report the synthesis and biological evaluation of QACs based on imidazole derivative, N-benzylimidazole. The antimicrobial activity was tested against a range of pathogenic bacteria and fungi, both ATCC and clinical isolates, showing varying activities ranging in minimal inhibitory concentrations (MICs) from as low as 7 ng/mL. The most promising compound, N-tetradecyl derivative (BnI-14), proved to be very potent against bacterial biofilms, even at sub-MIC doses, suggesting interference with the bacterial growth and/or division process. The BnI-14 treatment induces bacterial membrane disruption, as observed by fluorescence spectroscopy and atomic force microscopy and it also binds to DNA indicating that bacterial membrane might not be the only cellular target of QACs. Most importantly, BnI-14 exhibits low toxicity to healthy human cell lines, suggesting that N-benzylimidazolium-based QACs may be promising new antimicrobial agents.

In the last two decades, accumulating evidence pointed to the importance of autophagy in various human diseases. As an essential evolutionary catabolic process of cytoplasmatic component digestion, it is generally believed that modulating autophagic activity, through targeting specific regulatory actors in the core autophagy machinery, may impact disease processes. Both autophagy upregulation and downregulation have been found in cancers, suggesting its dual oncogenic and tumor suppressor properties during malignant transformation. Identification of the key autophagy targets is essential for the development of new therapeutic agents. Despite this great potential, no therapies are currently available that specifically focus on autophagy modulation. Although drugs like rapamycin, chloroquine, hydroxychloroquine, and others act as autophagy modulators, they were not originally developed for this purpose. Thus, autophagy may represent a new and promising pharmacologic target for future drug development and therapeutic applications in human diseases. Here, we summarize our current knowledge in regard to the interplay between autophagy and malignancy in the most significant tumor types: pancreatic, breast, hepatocellular, colorectal, and lung cancer, which have been studied in respect to autophagy manipulation as a promising therapeutic strategy. Finally, we present an overview of the most recent advances in therapeutic strategies involving autophagy modulators in cancer.

Pseudomonas aeruginosa is one the most common multidrug-resistant pathogens worldwide. It has been previously detected in marine shellfish, but its antibiotic resistance in such environment has not been explored. By combining PCR detection of acquired genes, and resistance-nodulation-cell division (RND) efflux studying, we investigated the multifactorial resistance traits of 108 P. aeruginosa isolates recovered from wild-growing Mediterranean mussels (Mytilus galloprovincialis) in Croatia. Eleven different resistance profiles were found, with the main mechanism being the overexpression of intrinsic efflux pump(s), particularly MexAB-OprM. Several acquired resistance determinants were detected, including the β-lactamase gene blaTEM-116, sulfamethoxazole resistance gene sul1, and the class 1 integron gene cassette carrying the streptomycin resistance gene aadA7. This study evidenced the multiple resistance in P. aeruginosa in shellfish from human-impacted marine environment, pointing to the underestimated role of the marine habitat for maintenance of multiresistant P. aeruginosa and, consequently, the potential risk for human and environmental health.

Nonribosomal peptide synthetases (NRPS) are multifunctional proteins that catalyze the synthesis of the peptide products with enormous biological potential. The process of biosynthesis starts with the adenylation (A) domain, which during the catalytic cycle undergoes extensive structural rearrangements. In this paper, we present the first study of the tyrocidine synthetase 1 A-domain (TycA- A) fluorescence properties. The TycA-A protein contains five potentially fluorescent Trp residues at positions 227, 301, 323, 376 and 406. The contribution of each Trp to the TycA-A emission was determined using protein variants bearing single Trp to Phe substitutions. The accessibility of the Trp side chains during adenylation showed that only W227 is affected by substrate binding. The protein variant containing solely fluorescent W227 residue was constructed and further used as a probe to explore the binding effect of different non-cognate amino acid substrates. The results indicate a different accessibility of W227 residue in the presence of non- cognate amino acids, which might offer an explanation for the higher aminoacyl-adenenylate leakage. Overall, our results suggest that intrinsic tryptophan fluorescence could be used as a method to probe the effect of substrate binding on the local structure in NRPS adenylation domains.

Mitochondria are organelles with numerous vital roles in cellular metabolism. Impaired or damaged mitochondria are degraded in autophagolysosomes in a process known as mitophagy. Given the fundamental role of mitophagy in maintenance of cellular homeostasis, methods and techniques with which to study this process are constantly evolving and emerging. So far, mitophagy flux was mostly monitored using fluorescently labeled LC3 protein on autophagosomal membrane and any of the labeled outer mitochondrial membrane proteins. However, this method is labor intensive, time consuming, and difficult to quantitatively validate due to the rapid mitochondrial turnover. Here, we describe a flow cytometry as a novel and promising quantitative method to monitor Bnip3- and Bnip3L/Nix-mediated mitophagy.

Here, we report a comparative study of the phytochemical profile and the biological activity of two onion extracts, namely Allium cepa L. and Allium × cornutum (Clementi ex Visiani 1842), members of the family Amaryllidaceae. The identification of flavonoids and anthocyanins, and their individual quantities, was determined by high-performance liquid chromatography (HPLC). The potency of both extracts to scavenge free radicals was determined by the DPPH (2,2'-diphenyl-1-picrylhydrazyl) radical-scavenging activity and oxygen radical absorbance capacity (ORAC) methods. The DNA protective role was further tested by the single-cell gel electrophoresis (COMET) assay and by Fenton's reagent causing double-strand breaks on the closed circular high copy pUC19 plasmid isolated from Escherichia coli. In the presence of both extracts, a significant decrease in DNA damage was observed, which indicates a protective role of Allium cepa and Allium × cornutum on DNA strand breaks. Additionally, cytotoxicity was tested on glioblastoma and breast cancer cell lines. The results showed that both extracts had antiproliferative effects, but the most prominent decrease in cellular growth was observed in glioblastoma cells.

Here we report a comparative study of the pyhtochemical profile and a biological activity of the two onion extracts, namely A. cepa L. and A.× cornutum (Clementi ex Visiani 1842). Identification of flavonoids and anthocyanins and their individual quantities were determined by high performance liquid chromatography (HPLC). The potency of both extracts to scavenge free radicals was determined by DPPH (2,2'-diphenyl-1-picrylhydrazyl) radical-scavenging activity and oxygen radical absorbance capacity (ORAC) methods. DNA protective role was further tested by single-cell gel electrophoresis (COMET) assay and by Fenton’s reagent causing double strand brakes on pUC19 plasmid. In the presence of both extracts, a significant decrease in the DNA damage was observed, which indicates a protective role of A. cepa and A.× cornutum on the DNA strand brakes. Additionally, cytotoxicity was tested on glioblastoma and breast cancer cell lines. The results showed that both extracts had antiproliferative effect, but the most prominent decrease in cellular growth was observed with glioblastoma cells

The mitophagy receptor Nix interacts with LC3/GABARAP proteins, targeting mitochondria into autophagosomes for degradation. Here we present evidence for phosphorylation-driven regulation of the Nix:LC3B interaction. Isothermal titration calorimetry and NMR indicate a ~100 fold enhanced affinity of the serine 34/35-phosphorylated Nix LC3-interacting region (LIR) to LC3B and formation of a very rigid complex compared to the non-phosphorylated sequence. Moreover, the crystal structure of LC3B in complex with the Nix LIR peptide containing glutamic acids as phosphomimetic residues and NMR experiments revealed that LIR phosphorylation stabilizes the Nix:LC3B complex via formation of two additional hydrogen bonds between phosphorylated serines of Nix LIR and Arg11, Lys49 and Lys51 in LC3B. Substitution of Lys51 to Ala in LC3B abrogates binding of a phosphomimetic Nix mutant. Functionally, serine 34/35 phosphorylation enhances autophagosome recruitment to mitochondria in HeLa cells. Together, this study provides cellular, biochemical and biophysical evidence that phosphorylation of the LIR domain of Nix enhances mitophagy receptor engagement.

Two series of novel ammonium salts containing the quinuclidine moiety were prepared in order to evaluate their antioxidative, antibacterial and antifungal potential. The synthesized homologues of 3-hydroxy (QOH) and 3-chloroquinuclidine (QCl) with the different N-benzyl substituents at the para-position (bromo, chloro or nitro group) were obtained in very good yields and characterized by IR and NMR spectroscopies and elemental analysis. All compounds were tested for antioxidative activity using the oxygen radical absorbance capacity (ORAC) assay and among tested samples, N-p-nitrobenzyl-3-hydroxyquinuclidinium bromide (QOH-4) exhibited the highest antioxidative potential (293.80 nmol (TE) mL-1), which was further investigated by the DNA nicking assay. The biological activity of selected compounds was evaluated by measuring the zone of inhibition and by determining the minimal inhibitory concentration (MIC) against three Gram-positive bacteria (B. cereus, E. faecalis and S. aureus), three Gram-negative bacteria (E. coli, P. aeruginosa and C. sakazakii) and three fungi species (C. albicans, A. niger and P. notatum). The bioactivity assay showed that some newly synthetized quaternary quinuclidinium compounds display a comparable or even better antibacterial and antifungal activity than the reference drugs such as gentamicin (GEN), cefotaxime (CTX) and amphotericin B (AMPHB). Among the tested compounds, N-p-chlorobenzyl-3-hydroxyquinuclidinium bromide (QOH-3) exhibited a considerable antibacterial efficiency against P. aeruginosa (MIC=0.39 µg mL-1) and QOH-4 displayed a potent antifungal activity against C. albicans (MIC=1.56 µg mL-1).

Mitochondria are organelles with numerous vital roles in cellular metabolism. Impaired or damaged mitochondria are degraded in autophagolysosomes in a process known as mitophagy. Given the fundamental role of mitophagy in maintenance of cellular homeostasis, methods and techniques with which to study this process are constantly evolving and emerging. So far, mitophagy flux was mostly monitored using fluorescently labeled LC3 protein on autophagosomal membrane and any of the labeled outer mitochondrial membrane proteins. However, this method is labor intensive, time consuming, and difficult to quantitatively validate due to the rapid mitochondrial turnover. Here, we describe a flow cytometry as a novel and promising quantitative method to monitor Bnip3- and Bnip3L/Nix-mediated mitophagy.

Pro-inflammatory cytokines play an important role in teleost defence against numerous types of pathogens, therefore are often used as biomarkers during various infections. In order to evaluate Atlantic bluefin tuna IL-1β, TNFα1 and TNFα2 induction by PAMPs, we quantified their expression during in vitro stimulation of peripheral blood leukocytes with LPS, Poly I:C. Furthermore, their role in acute and chronic parasitic infection was examined during natural infection of Pseudocycnus appendiculatus (Copepoda) and Didymosulcus katsuwonicola (Digenea), as well as during leukocyte exposure to total protein extracts isolated from two parasite species. Induction of ABT IL- 1β and TNFα2 by PAMPs and protein extracts from D. katsuwonicola and P. appendiculatus, as well as during natural infection with two parasites, suggests these cytokines play an important role in inflammation, being engaged in controlling parasite infections, in contrast to ABT TNFα1. Cellular innate response to the digenean D. katsuwonicola showed rather chronic character, resulting with parasite encapsulation in connective tissue. Mast cells, eosinophils, goblet cells, and occasional rodlet cells found at the site of infection, along with the induction of TNFα2, suggest the presence of a moderate inflammatory reaction that fails to seriously endanger digenean existence. In contrast, copepod P. appendiculatus, attached to the gill epithelium by clamping, caused direct tissue disruption with undergoing necrotic or apoptotic processes, and extensive proliferation of rodlet and goblet cells. Differential expression patterns of target cytokines in tissue surrounding two parasites and in vitro PBL model suggest that quality and quantity of tuna immune response is conditioned by parasite adaptive mechanisms and pathogenicity.

Mutations, replacing amino acids involved in the formation of hydrogen bonds between subunits of dimeric alkaline phosphatase, have been prepared. Besides establishing its influence on kinetic properties and structural stability, alterations in protein dynamic properties have been studied using room temperature phosphorescence. Kinetic properties of mutant enzymes were virtually the same, differing from the wild type enzyme in the kcat value that was almost twice lower. Changes in rigidity of the protein region of interest was studied by measuring Trp109 phosphoresce lifetime and the rate of its phosphorescence quenching with acrylamide. Changes in protein dynamic properties of mutant proteins compared to the wild type enzyme, as indicated by RTP, did not parallel changes in kinetic properties suggesting that an entropy effect is not responsible for the reduction of kinetic properties. Instead, combined kinetic and dynamic consequences of mutations breaking the hydrogen bonds suggest that breaking of specific links, involved in transmission of conformational change, could be responsible for altered kinetic properties.

In plant tissues, enzymes implicated in the lipoxygenase (LOX) pathway are responsible for the hydroperoxydation of polyunsaturated fatty acids, ultimately leading to the production of small chemical species involved in several physiological processes. During industrial olive oil production, these enzymes are activated upon crushing and grinding of olive fruit tissue, subsequently leading to the synthesis of volatile compounds responsible for the positive aroma and flavor of the oil. An investigation of LOX activity during olive fruit ripening and malaxation could assist in the production of oils with favorable aroma and taste. Therefore, a reliable method for olive LOX purification is crucial. Here we report a critical review of six LOX extraction protocols, two of which have shown minimum enzyme activity, possibly leading to misconceptions in the interpretation of experimental data. Future research concerning olive LOX should employ extraction methods that preserve enzyme activity.

The adenylation (A) domain in nonribosomal peptide synthetases catalyses a two-step reaction in which an amino acid is activated and then transferred to the neighbouring thiolation (T) domain. In this study, we investigated the role of the conserved A9 core sequence of the A-domain of tyrocidine synthetase 1, by analysis of single amino acid mutations in the A9 region. Mutation of an absolutely conserved proline (P490G) significantly reduced the conformational stability of the protein, as evidenced by increased susceptibility to proteolytic cleavage and denaturation. All mutant A-domains were capable of amino acid activation, but the activity in the overall reaction was reduced. Surprisingly, the S491R mutant (mutation at the first residue following the A9 motif) showed elevated overall activity compared to the wild-type protein. Our results suggest that the A9 core sequence plays a role in the second reaction step, in which it could serve as a "clip" for the proper positioning of residues important for the interaction with the T-domain, and/or stabilisation of the thioester-forming conformation.

During an environmental study of bacterial resistance to antibiotics in coastal waters of the Kaštela Bay, Adriatic Sea, Croatia, 47 Burkholderia cepacia complex (Bcc) isolates were recovered from seawater and mussel (Mytilus galloprovincialis) samples. All isolates showed multiple antibiotic resistance. Among the isolates, two Burkholderia cenocepacia isolates produced chromosomally encoded TEM-116 extended-spectrum β-lactamase (ESBL). Analysis of outer membrane proteins revealed that decreased expression of a 36-kDa protein could be associated with a high level of β-lactam resistance in both isolates. Phenotypic study of efflux system also indicated an over-expression of Resistance-Nodulation-Cell Division (RND) efflux-mediated mechanism in one of the isolates. This study demonstrated the presence of Bcc in seawater and M. galloprovincialis, which gives evidence that coastal marine environment, including mussels, could be considered as a reservoir for Bcc species. Detection of ESBL-encoding genes indicates the potential role of these bacteria in the maintenance and dispersion of antibiotic resistance genes.