Extracellular proteinases in natural isolates of Staphylococci

U ovom radu su detaljnije biohemijski okarakterisane proteinaze prirodnih izolata Staphylococcus sp. F22, F86, M104, S2007 i S2105. Utvrđeno je da se radi o proteinazama relativno male molekulske mase (od 20 do 32 kDa) koje spadaju u prave ekstracelularne enzime, pošto se sa površine ćelije odvajaju u medijum za rast. Temperaturni optimumi ovih proteinaza se kreću od 30 do 37°C, a pH optimumi u opsegu od 6,5 do 8. Joni bakra inhibiraju njihovu aktivnost, dok joni kalcijuma stimulišu aktivnost proteinaza iz izolata F22 M104 i S2007. Pored kazeinskih frakcija, proteinaze stafilokoka hidrolizuju i druge proteinske supstrate, kao što su želatin i BSA. U eksperimentima sa proteinaznim inhibitorima utvrđeno je da proteinaze izolata F22 i M104 pripadaju serinskoj klasi, proteinaze S2007 i S2105 klasi metaloproteinaza dok proteinazu izolata F86 na ovaj način nije bilo moguće precizno klasifikovati.Biochemical characteristics of proteinases from natural isolates of Staphylococcus sp. F22, F86, M104, S2007 and S2105 have been studied. It was found that these proteinases have relatively low molecular masses (from 20 to 32 kDa), and that they are released from the cell envelope in to the growth medium. Their temperature optima are between 30 and 37°C and their pH optima range from 6,5 to 8. Copper ions inhibit their activity, but the presence of calcium ions stimulates the activity of proteinases from isolates F22, M104 and S2007. Beside casein fractions, they also hydrolyze heterologous protein substrates, such as BSA and gelatin. Experiments with specific proteinase inhibitors revealed that proteinases from isolates F22 and M104 belong to the serine group of proteinases, S2007 and S2105 proteinases were classified as metalloproteinases. Type of F86 proteinase in these experiments could not be clearly determinated

Post-translational regulation of the RpoS and PsrA genes in pseudomonas putida WCS358: The role of ClpXP protease

RpoS i PsrA proteini su ključni transkripcioni regulatori koji se kod pseudomonada aktiviraju u odgovoru na stacionarnu fazu rasta. Cilj ove studije bio je utvrđivanje uloge ClpXP (ATP zavisna serin proteaza) u stabilnosti RpoS i PsrA proteina tokom različitih faza rasta u Pseudomonas putida WCS358. "Western blot" analiza proteinskih ekstrakata P. putida WCS358 i P. putida WCS358 clpX::Km iz rane eksponencijalne, Kasne eksponencijalne i stacionarne faze rasta, sa antitelima na RpoS i PsrA, pokazala je da ClpXP degraduje RpoS i PsrA u ranoj eksponencijalnoj fazi rasta.The RpoS and PsrA proteins are key transcriptional regulators that are activated in response to the stationary phase of growth in pseudomonads. This study was designed to establish whether ClpXP (ATP-dependent serine protease) regulates levels of RpoS and PsrA in Pseudomonas putida WCS358. Western blot analysis of P. putida WCS358 protein extracts from the early exponentianl, late exponential, and stationary phases of growth with antibodies against RpoS and PsrA revealed that these proteins are degraded by ClpXP in the early exponential phase of growth. The obtained results demonstrate a role for ClpXP protease in post-translational regulation of proteins encoded by the rpoS and psrA genes in Pseudomonas spp

Characterization of lactococci isolated from homemade kefir

Pet izolata laktokoka proizvođača bakteriocina izolovanih iz kefira pripremljenog na tradicionalan način determinisano je kao Lactococcus lactis subsp. lactis. Analizirani izolati poseduju različite plazmidne profile i među njima nije detektovana uzajamna antimikrobna aktivnost. Takođe prirodni izolat BGKF26 je rezistentan na aktivnost soja NP45, proizvođača nizina. Eksperimenti čišćenja plazmida pokazali su da se geni _a sintezu bakteriocina i proteinaza nalaze na različitim genetičkim elementima, osim kod izolata BGKF26. Sinteza analiziranih bakteriocina zavisi od koncentracije kazitona ili triptona u medijumu. Više koncentracije kazitona ili triptona indukuju bakteriocinsku aktivnost. Hibridizacioni eksperimenti su pokazali da su analizirani bakteriocini najverovatnije slični laktokokcinima.Five bacteriocin-producing lactococci isolates from traditionally prepared kefir were determined as Lactococcus lactis subsp. lactis. The analyzed isolates showed different plasmid profiles and no cross inhibition between them was detected. Moreover, natural isolate BGKF26 was resistant to the antimicrobial activity of nisin producing strain NP45. Plasmid curing experiments revealed that the genes encoding bacteriocin and proteinase production are located on separate genetic elements, except in BGKF26. Production of the tested bacteriocins depends on the concentration of casitone or triptone in the medium. Higher concentrations of casitone or triptone induce bacteriocin activity. Our DNA-DNA hybridization analyses suggest that the analyzed antimicrobial compounds probably are lactococcin-like bacteriocins

Effect of methionine and cysteine deprivation on growth of different natural isolates of Lactobacillus spp. in chemically defined media

Cilj ovog rada je bio da se utvrdi sposobnost prirodnih izolata laktobacila,izolovanih iz različitih ekoloških niša da rastu u hemijski definisanom medijumu sa ili bez prisustva aminokiselina koje sadrže sumpor, metionin i/ili cistein. Dobijeni rezultati su pokazali da je esencijalna aminokiselina za rast izolata L. paracasei subsp. paracasei BGHN14 i BGSJ2-8 cistein, dok je za izolate BGHN40, BGCG31, BGHV54T, koji pripadaju vrsti L. plantarum-metionin. Metionin je esencijalna aminokiselina za rast soja L. rhamnosus BGHV58T. Ostali analizirani sojevi,kao što su L. plantarum BGSJ3-18,BGZB19,BGHV52Ta i BGHV43T za svoj rast zahtevaju prisustvo obe aminokiseline u medijumu.The purpose of this study was to determine the ability of natural isolates of lactobacilli from different ecological niches to grow in a chemically defined medium in the presence or absence of sulphur-containing amino acids, methionine and/or cysteine. The obtained results indicate that cysteine is essential for growth of L. paracasei subsp. paracasei BGHN14 and BGSJ2-8, while methionine is essential for isolates BGHN40, BGCG31, and BGHV54T of the species L. plantarum. Methionine is also essential for growth of L. rhamnosus BGHV58T. Other analyzed strains, such as L. plantarum BGSJ3-18, BGZB19, BGHV52Ta, and BGHV43T, require the presence of both amino acids for their growth

Dynamics of sodium dodecyl sulfate utilization andantibiotic susceptibility of strain Pseudomonas sp. ATCC19151

Poznato je da soj Pseudomonas sp. ATCC19151 poseduje gen koji kodira Potencijalnu alkilsulfatazu. U ovom radu analizirana je sposobnost rasta ovog soja u minimalnom medijum usa različitim koncentracijama natrijum dodecilsulfata (0.5, 0.75 i 1 %) kao jedinim izvorom ugljenika. Pokazano je da Pseudomonas sp. ATCC19151 ispoljava najbolji rast uminimalnom medijumusa 0.5 % natrijum dodecil sulfata, te je stoga ova koncentracija uzeta kao optimalna za testiranje dinamike korišćenja natrijum dodecil sulfata tokom različitih faza rasta. Dinamika korišćenja natrijum dodecil sulfata podudarala se sa rastom kulture. Pored toga u cilju detaljnije karakterizacije soja, analizirana je i osetljivost Pseudomonas sp. ATCC19151 na antibiotike. Pokazano je da je analizirani soj rezistentan na šest (ampicilin, tetraciklin, hloramfenikol, tobramicin, nalidiksičnukiselinui gentamicin) od devet analiziranih antibiotika.Pseudomonas sp. ATCC19151 harbors a gene encoding a putative alkylsulfatase (sdsA). Here we report a growth ability of this strain in minimal media containing 0.5, 0.75, and 1% sodium dodecyl sulfate as the sole carbon source. The most prominent growth was detected for the minimal medium with 0.5% SDS, so this concentration of SDS was used to monitor Pseudomonas sp. ATCC19151 SDS biodegradation dynamics. Bacterial growth coincided with the disappearance of SDS. Antibiotic susceptibility was tested as well. Pseudomonas sp. ATCC19151 was resistant to six out of nine tested antibiotics, including ampicillin, tetracycline, chloramphenicol, tobramycin, nalidixic acid, and gentamycin

Influence of carbohydrates on cell properties of Lactobacillus rhamnosus

Lactobacilli represent normal commensals of the human body, particularly in the gut and vagina where they protect these environments from incoming pathogens via a variety of mechanisms. The influence of the carbohydrate source present in reconstituted MRS growth medium on the different cell properties of two Lactobacillus rhamnosus strains were examined. Two human vaginal isolates, BGHV719 and exopolysaccharide producer strain BGHV954 were analyzed. The results demonstrated that unlike in reconstituted MRS with glucose as a carbon source, the presence of fructose, mannose, or rhamnose, significantly reduced cell surface hydrophobicity of both strains. In addition, differences in cell wall protein composition of L. rhamnosus BGHV719 and alterations in colony mucoidity of L. rhamnosus BGHV954 were also demonstrated. Light and SEM microscopy revealed differences on the cellular level when BGHV719 was cultivated in the presence of different sugars. The results of this study point out the importance of complex relationships between growth medium composition and the different aspects of bacterial behavior, and call for more detailed analyses of versatile bacterial responses to the changes in the environment, including vaginal ecosystem. This is especially important since lactobacilli are amongst the most widely used of probiotics

Lactobacillus fermentum Postbiotic-induced Autophagy as Potential Approach for Treatment of Acetaminophen Hepatotoxicity

The aim of this study was to investigate the potential of postbiotics originated from Lactobacillus fermentum BGHV110 strain (HV110) to counteract acetaminophen (APAP)-induced hepatotoxicity in HepG2 cells. This strain was selected according to its autophagy inducing potential, based on previous studies reporting protective role of autophagy in APAP caused cellular damage. Cell viability was assessed using MTT and LDH assays, while autophagy was monitored by qPCR analysis of BECN1, Atg5, p62/SQSTM1, and PINK1 mRNA expression and by Western blot analysis of p62/SQSTM1 and lipidated LC3 accumulation. Our results showed that detrimental effect of APAP on cell viability was suppressed in the presence of HV110 which was linked with increased conversion of LC3 protein and p62/SQSTM1 protein degradation. Additionally, higher p62/SQSTM1 and PINK1 mRNA transcription were noticed in cells co-treated with APAP/HV110, simultaneously. In conclusion, this study suggests that HV110 enhances activation of PINK1-dependent autophagy in HepG2 cells and its eventual co-supplementation with APAP could be potentially used for alleviation of hepatotoxic side effects caused by APAP overdose

Analysis of natural isolates of Lactobacilli resistant to bacteriocin nisin

Kolekcija bakterija mlečne kiseline (BMK) je napravljena od mikroorganizama izolovanih iz fermentisanih mlečno-kiselinskih proizvoda dobijenih na tradicionalan način. Iz kolekcije 51 izolat je identifikovan kao Lactobacillus sp. Svi izolovani laktobacili pripadaju grupi mezofilnih sojeva koji dobro rastu na temperaturama od 15°C i 30°C, a ne rastu na temperaturi od 45°C. Testiranje sposobnosti rasta u prisustvu nizina pokazalo je da su izolati BGCGK4, BGHN40, BGBUK2-8, BGBUK2-7 i BGBUK2-16 rezistentni na bakteriocin nizin. U eksperimentu određivanja minimalne inhibitorne koncentracije (MIK) za nizin pokazano je da je najrezistentniji izolat Lactobacillus sp. BGCGK4. Izolat BGBUK2-16, determinisan kao Lactobacillus paracasei subsp. paracasei, produkuje bakteriocin označen kao Bac217 i pokazuje rezistenciju na 8000 IU/ml. Čišćenjem plazmida iz soja BGBUK2-16 dobijena su 2 derivata označena kao BGBUK2-16/K2 i BGBUK2-16/K4. Derivat BGBUK2-16/K2 zadržao je rezistenciju na Bac217 i nizin, ali je izgubio sposobnost sinteze Bac217, dok je derivat BGBUK2-16/K4 pored gubitka sposobnosti sinteze Bac217 postao senzitivan na Bac217 i nizin. Prirodno rezistentni laktobacili se mogu iskoristiti za pripremanje starter kultura u kombinaciji sa nizinom kao konzervansom u cilju kontrolisane mlečno-kiselinske fermentacije.The collection of lactic acid bacteria (LAB) was made by isolation of microorganisms from fermented products traditionally manufactured in different geographical regions (high mountains, river valleys, seaside, etc). Among collected LAB, 51 isolates were identified as Lactobacillus sp. Results showed that all isolated lactobacilli were mesophilic strains, since they grew at 15°C and 30°C but not at 45°C. Testing the ability of isolated lactobacilli to grow in the presence of nisin revealed that Lactobacillus sp. isolates designed BGCGK4, BGHN40, BGBUK2-8, BGBUK2-7 and BGBUK2-16 were resistant to nisin. Determination of the minimal inhibitory concentrations (MIC) for nisin revealed that the most resistant isolate was Lactobacillus sp. BGCGK4. Isolate BGBUK2-16, determined as Lactobacillus paracasei subsp. paracasei, produces bacteriocin, named Bac217 and showed a resistance to 8000 IU/ml of nisin. Plasmid curing of BGBUK2-16 resulted in derivatives BGBUK2-16/K2 and BGBUK2-16/K4. Derivative BGBUK2-16/K2 retained resistance to Bac217 and nisin, but lost the ability to synthesise Bac217. Derivative BGBUK2-16/K4 lost concomitantly the resistance to both Bac217 and nisin

On Gergonne point of the triangle in isotropic plane

Using the standard position of the allowable triangle in the isotropic plane relationships between this triangle and its contact and tangential triangle are studied. Thereby different properties of the symmedian center, the Gergonne point, the Lemoine line and the de Longchamps line of these triangles are obtained