Molecular analysis of enterolysin A and entL gene cluster from natural isolate Enterococcus faecalis BGPT1-10P

Soj Enterococcus faecalis BGPT1-10P je izolovan iz domaćeg polutvrdog sira, poreklom sa Stare Planine. Rezultati pokazuju da soj BGPT1-10P sintetiše termolabilan bakteriocin, enterolizin A, sa širokim spektrom delovanja, uključujući patogene bakterije roda Listeria i Candida. EntL gen, odgovoran za sintezu ovog bakteriocina, je hromozomalno lokalizovan. Analiza nukleotidne sekvence entL gena kod prirodnog izolata En. faecalis BGPT1-10P je identična sa entL genom soja En. faecalis LMG 2333, koji je prethodno okarakterisan. Pokazana je jedinstvena sekvenca entL gena i njegove okoline, koju čine orf1, orf2 i orf3 geni, kao i scpE gen. Prvi put je kod prirodnog izolata okarakterisan scpE gen, koji kodira virulentni faktor stafopain peptidazu. Funkcionalna analiza entL gena je pokazala da je kompletna genetička informacija, neophodna za sintezu i aktivnost enterolizina A, sadržana u entL genu. Soj BGPT1-10P osim enterolizina, sintetiše i želatinazu i citolizin i sadrži set različitih virulentnih faktora. Pored toga, BGPT1-10P nosi ermB i tetM gene, odgovorne za rezistenciju na eritromicin i tetraciklin.Strain Enterococcus faecalis BGPT1-10P was isolated from artisanal semi-hard homemade cheese from Stara Planina, Serbia. Results showed that BGPT1-10P synthesized a heat labile bacteriocin with a broad spectrum of activity, including Listeria and Candida species. Further analysis revealed that synthesized bacteriocin is enterolysin A. Moreover, the entL gene encoding enterolysin A was found to be located on the chromosome. The entL gene was cloned and sequenced. Analysis of nucleotide sequence showed that the entL gene in natural isolate En. faecalis BGPT1-10P is identical to that of the entL gene described previously in En. faecalis LMG 2333. Within the cloned DNA fragment containing the entL gene, four ORFs were detected. One of them was identified as the scpE gene, which encodes a virulent factor staphopain peptidase. Functional analysis of the entL gene showed that the complete genetic information necessary for the synthesis of enterolysin A were directly linked solely to it. Strain BGPT1-10P also synthesized gelatinase and citolysin, and contained a set of virulent factors. In addition, BGPT1-10P carries the ermB and tetM genes conferring the resistance to erythromycin and tetracycline, respectively

Molecular analysis of enterolysin a and entL gene cluster from natural isolate Enterococcus faecalis BGPT1-10P

Strain Enterococcus faecalis BGPT1-10P was isolated from artisanal semi-hard homemade cheese from Stara Planina, Serbia. Results showed that BGPT1-10P synthesized a heat labile bacteriocin with a broad spectrum of activity, including Listeria and Candida species. Further analysis revealed that synthesized bacteriocin is enterolysin A. Moreover, the entL gene encoding enterolysin A was found to be located on the chromosome. The entL gene was cloned and sequenced. Analysis of nucleotide sequence showed that the entL gene in natural isolate En. faecalis BGPT1-10P is identical to that of the entL gene described previously in En. faecalis LMG 2333. Within the cloned DNA fragment containing the entL gene, four ORFs were detected. One of them was identified as the scpE gene, which encodes a virulent factor staphopain peptidase. Functional analysis of the entL gene showed that the complete genetic information necessary for the synthesis of enterolysin A were directly linked solely to it. Strain BGPT1-10P also synthesized gelatinase and citolysin, and contained a set of virulent factors. In addition, BGPT1-10P carries the ermB and tetM genes conferring the resistance to erythromycin and tetracycline, respectively. [Projekat Ministarstva nauke Republike Srbije, br. 173019

Značaj funkcionalne anatomije u istraživanjima transporta vode i asimilata u plodove u razvoju

The yield of crop plants and fruit growth depends, to a large extent, on the efficiency of the vascular tissue, in optimal conditions, as well as in stress conditions. Most of the material on which fruit growth depends is transported from the stem into the fruit through the fruit pedicel, by the xylem and phloem, so the anatomy of fruit pedicel is an important factor in understanding water transport from stem to fruit. This paper provides an overview of micro-morphological research of the tomato fruit pedicel using various methods in the Laboratory for Functional Anatomy of Crop Plants at the Faculty of Agriculture, Belgrade University. Such an approach is important for understanding transport mechanisms as important physiological processes occurring during fruit growth.Prinos gajenih biljaka i rast plodova u velikoj meri zavisi od efikasnosti provodnog sistema, kao u optimalnim uslovima, tako i u uslovima vodnog deficita. Većina materija od kojih zavisi rast plodova, transportuje se u plod kroz peteljku ploda ksilemom i floemom, pa je tako anatomija peteljke važan faktor za razumevanje transporta vode i asimilata. U ovom radu dat je pregled mikromorfoloških istraživanja peteljki plodova paradajza primenom različitih metoda. Ovakav pristup je važan za razumevanje transportnih mehanizama kao važnih činilaca fizioloških procesa značajnih za rast i sazrevanje ploda

Enterococci from Raw-Milk Cheeses: Current Knowledge on Safety, Technological, and Probiotic Concerns

The present study is focused on the safety, technological characteristics, and probiotic evaluation of Enterococcus species from different artisanal raw milk dairy products, mainly cheeses with ripening. Apart from proteolytic and lipolytic activities, most enterococci show the ability to metabolize citrate and convert it to various aromatic compounds. Long-ripened cheeses therefore have a specific flavor that makes them different from cheeses produced from thermally treated milk with commercial starter cultures. In addition, enterococci are producers of bacteriocins effective against spoilage and pathogenic bacteria, so they can be used as food preservatives. However, the use of enterococci in the dairy industry should be approached with caution. Although originating from food, enterococci strains may carry various virulence factors and antibiotic-resistance genes and can have many adverse effects on human health. Still, despite their controversial status, the use of enterococci in the food industry is not strictly regulated since the existence of these so-called desirable and undesirable traits in enterococci is a strain-dependent characteristic. To be specific, the results of many studies showed that there are some enterococci strains that are safe for use as starter cultures or as probiotics since they do not carry virulence factors and antibiotic-resistance genes. These strains even exhibit strong health-promoting effects such as stimulation of the immune response, anti-inflammatory activity, hypocholesterolemic action, and usefulness in prevention/treatment of some diseases

Analysis of the lactic acid bacteria microflora in traditional Caucasus cow's milk cheeses

A total of 157 lactic acid bacteria (LAB) were isolated from three hand-made cheeses taken from different households in the region of the Caucasus Mountains. The cheeses were manufactured from cow's milk without the addition of a starter culture. The isolates of LAB were characterized by subjecting them to phenotypic and genotypic tests. The results of identification of LAB indicate that the examined cheeses contained 10 species, viz., Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus arizonensis, Lactobacillus farciminis, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides subsp. mesenteroides, Leuconostoc pseudomesenteroides, Enterococcus faecium, and Enterococcus faecalis. The strains within the species L. plantarum, L. arizonensis, L. paraplantarum, L. farciminis, and L. pseudomesenteroides showed good proteolytic activity

Analysis of the lactic acid bacteria microflora in traditional Caucasus cow's milk cheeses

Iz tri ručno pravljena sira uzeta iz različitih domaćinstava smeštenih u regionu planine Kavkaz, izolovano je ukupno 157 bakterija mleč ne kiseline (LAB). Sirevi su pravljeni od kravljeg mleka bez dodatka starter kulture. Izolati LAB su okarakterisani fenotipskim i genotipskim testovima. Rezultati identifikacije LAB pokazuju da je u ispitivanim sirevima prisutno deset vrsta, kao što su: Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus arizonensis, Lactobacillus farciminis, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides subsp. mesenteroides, Leuconostoc pseudomesenteroides, Enterococcus faecium i Enterococcus faecalis. Sojevi u okviru vrsta L. plantarum, L. arizonensis, L. paraplantarum, L. farciminis i L. pseudomesenteroides su pokazivali dobru proteolitičku aktivnost.A total of 157 lactic acid bacteria (LAB) were isolated from three hand-made cheeses taken from different households in the region of the Caucasus Mountains. The cheeses were manufactured from cow's milk without the addition of a starter culture. The isolates of LAB were characterized by subjecting them to phenotypic and genotypic tests. The results of identification of LAB indicate that the examined cheeses contained 10 species, viz., Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus arizonensis, Lactobacillus farciminis, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides subsp. mesenteroides, Leuconostoc pseudomesenteroides, Enterococcus faecium, and Enterococcus faecalis. The strains within the species L. plantarum, L. arizonensis, L. paraplantarum, L. farciminis, and L. pseudomesenteroides showed good proteolytic activity

Novel Aggregation Promoting Factor AggE Contributes to the Probiotic Properties of Enterococcus faecium BGGO9-28

The understanding of mechanisms of interactions between various bacterial cell surface proteins and host receptors has become imperative for the study of the health promoting features of probiotic enterococci. This study, for the first time, describes a novel enterococcal aggregation protein, AggE, from Enterococcus faeciurn BGGO9-28, selected from a laboratory collection of enterococcal isolates with auto aggregation phenotypes. Among them, En. faecium BGG09-28 showed the strongest auto -aggregation, adhesion to components of ECM and biofilm formation. Novel aggregation promoting factor AggE, a protein of 178.1 kDa, belongs to the collagen -binding superfamily of proteins and shares similar architecture with previously discovered aggregation factors from lactic acid bacteria (LAB). Its expression in heterologous enterococcal and lactococcal hosts demonstrates that the aggE gene is sufficient for cell aggregation. The derivatives carrying aggE exhibited the ten times higher adhesion ability to collagen and fibronectin, possess about two times higher adhesion to mucin and contribute to the increase of biofilm formation, comparing to the control strains. Analysis for the presence of virulence factors (cytolysin and gelatinase production), antibiotic resistance (antibiotic susceptibility) and genes (cylA, egg, gelE, esp, hyiN, ace, efaks, and efagn) showed that BGG09-28 was sensitive to all tested antibiotics, without hemolytic or gelatinase activity. This strain does not carry any of the tested genes encoding for known virulence factors. Results showed that BGGO9-28 was resistant to low pH and high concentrations of bile salts. Also, it adhered strongly to the Caco-2 human epithelial cell line. In conclusion, the results of this study indicate that the presence of AggE protein on the cell surface in enterococci is a desirable probiotic feature

Micromorphology of lettuce (Lactuca sativa L.) pollen grains

In order to contribute to palynological studies of cultivated plants in Serbia, micromorphological features of pollen grains of Lactuca sativa L. (Asteraceae) were examined by both, light microscopy (LM) and scanning electron microscopy (SEM). Morphological parameters, such as grain size and shape, apertures and exine ornamentation were described. The morphometric data such as length of polar axis (P), length of equatorial axis (E), P/E ratio, lacunae diameter, echinae size, and thickness of exine excluding echinae were noted. Pollen grain were found to be isopolar, radially symmetrical, medium-sized, oblate-spheroidal in shape (P/E = 0.94), sub-circular in equatorial view, and hexagonal in polar view with straight sides in outline. The length of the polar axis (P) is 32.6 ± 3.7 μm and of the equatorial diameter (E) 34.8 ± 2.7 μm. The pollen grain is 3-zonocolporate. Each compound aperture consists of ectoaperture which is a meridionally elongated colpus with rounded ends, and pore-shaped endoaperture. Exine ornamentation is echinolophate. Lophate (fenestrate) pollen grains, typical for Asteraceae, are characterized by number of lacunae that appear as large window-like spaces, a depressed area lacking ectexine. Each lacuna is surrounded by a system of echinate ridges. Lettuce pollen grains have 15 lacunae, 3 poral, 6 abporal and 6 paraporal (polar regions are without lacunae, small, reduced to triradiate ridge). The poral lacuna communicating with adjacent abporal lacunae via interlacunar gaps is observed rounded in shape. Paraporal lacunae, lying in the mesocolpial region adjacent to one side of an equatorial ridge, are pentagonal. Abporal lacunae are rounded or angular, broad towards the pole. Average lacuna diameter is 10.8 ± 2.5 μm. Lophae (ridges) are with one row of echines that are 1.9 ± 0.3 μm long, pointed and some are curved. The еctexine thickness excluding echines, measured under LM, averaged 5.7 ± 0.7 µm

Characterization of lactic acid bacteria isolated from artisanal Zlatar cheeses produced at two different geographical location

Iz belog polutvrdog zlatarskog sira, označenog kao BGNV, izolovan je osamdeset jedan soj bakterija mlečne kiseline (BMK). Sir je uzet iz jednog seoskog domaćinstva smeštenog na severnoj strani planine Zlatar. Zlatarski BGNV sir je napravljen od svežeg kravljeg mleka bez dodatka starter kulture. Svi izolati BMK su okarakterisani fenotipskim i genotipskim testovima. Identifikacija sojeva je rađena rep-PCR analizom sa (GTG)5 prajmerom i 16S rDNK sekvenciranjem. Najzastupljenije vrste u zlatarskom BGNV siru su bile Lactobacillus casei/paracasei (65.43%) i Enterococcus faecalis (29.63%. Dva fakultetivno anaerobna štapića su identifikovana kao Lactobacillus plantarum (2.47%), a dva obligatna heterofermentativna izolata BMK su identifikovani kao Lactobacillus parabuchneri (2.47%). Od svih 81 testiranih izolata, samo osam eneterokoka su bili proizvođači antimikrobnih komponenti. Četrnaest od 16 testiranih izolata laktobacila je pokazivalo srednju do vrlo dobru proteolitičku aktivnost. Svih 57 laktobacila iz zlatarskog BGNV sira veoma sporo grušaju mleko, ili ga uopšte ne grušaju. Međutim, tri izolata enterokoka, BGNV1-63, BGNV1-76 i BGNV1-80, su pokazivala vrlo dobru aktivnost u mleku i grušala su ga za 5 h. Ove enterokoke su pokazivale vrlo visoku proteolitičku aktivnost potpuno hidrolizirajući αs1- i κ- kazein nakon 3 h, a β-kazein nakon 30 min inkubacije. Pored toga, ova tri izolata enterokoka degradovala su želatin. Upoređujući dobijene rezultate sa onima prethodno dobijenim ispitivanjem BMK u drugom zlatarskom BGZLS siru, napravljenom takođe, od svežeg kravljeg mleka, može se zaključiti da je mikroflora BMK zlatraskog BGNV sira manje raznovrsna.Eighty-one strains of lactic acid bacteria (LAB) were isolated from white semi-hard homemade cheese, designated Zlatar BGNV, which was taken from household settled on Northern side of mountain Zlatar. The Zlatar BGNV cheese was manufactured from raw cow's milk without addition of the starter culture. All isolates of LAB were characterized by phenotypic and genotypic tests. Identification of strains was done by the repetitive extragenic palindromic-polymerase chain reaction (rep-PCR) with (GTG)5 primer and 16S rDNA sequence analysis. The most present species in Zlatar BGNV cheese were Lactobacillus casei/paracasei (65.43%) and Enterococcus faecalis (29.63%). Two facultative heterofermentative rods were identified as Lactobacillus plantarum (2.47%), and two obligate hetrofermentative LAB isolates as Lactobacillus parabuchneri (2.47%). Among all 81 tested isolates, only eight enterococci were producers of antimicrobial compounds. Fourteen of 16 tested lactobacilli isolates showed medium to very good proteolytic activity. All 57 lactobacilli from the Zlatar BGNV cheese curdled milk very slowly or did not curdle milk at all. However, three isolates of enterococci, BGNV1-63, BGNV1-76 and BGNV1-80, showed very good activity in milk and curdled milk within 5 h. They showed very high proteolytic activity hydrolyzing completely αs1- and κ-casein after 3 h, and β-casein after 30 min of incubation. In addition, those three enterococcal isolates degraded gelatin. Comparing obtained results with those previously achieved in examination of LAB microflora in another Zlatar BGZLS cheese made also from raw cow's milk, it can be concluded that LAB microflora in the Zlatar BGNV cheese is less diverse

Characterization of the bacteriocin-producing strain Lactobacillus paracasei subsp. paracasei BGUB9

The strain Lactobacillus paracasei subsp. paracasei BGUB9 that was isolated from traditionally homemade hard cheese produces bacteriocin designated as BacUB9, with an approximate molecular mass of 4 kDa. Biochemical characterization and the antimicrobial activity test of BacUB9 were performed. The onset of BacUB9 biosynthesis was observed at the end of an exponential phase of growth. Bacteriocin UB9 retained the antimicrobial activity within the pH range from 1 to 10 and after treatment at 100oC for 30 min. The bacteriocin is susceptible to the activity of proteolytic enzymes. Bacteriocin BacUB9 has a very narrow antimicrobial spectrum, limited to several strains that belong to closely related species. The effect of BGUB9 on the growth of the strain Lactobacillus paracasei subsp. paracasei BGHN14 in a mixed culture was monitored. The mode of action of BacUB9 on the strain BGHN14 was identified as bacteriostatic. Plasmid curing results indicated that a plasmid, designated as pUB9, seemed to be responsible for both bacteriocin BacUB9 production and host immunity