Provider: - Institution: - Data provided by Europeana Collections- A large number of Lactobacillus species play an important role in the production of fermented dairy products and meat products, and they are also present in fermented vegetables and grain products. Lactobacilli are used in medicine due to the positive effects that some strains have on the health of the host. Some Lactobacillus strains have a probiotic potential due to their ability to produce various antimicrobial compounds, exopolysaccharides (EPS), proteinases and other. Species of the Lactobacillus genus have the potential to inhibit the growth of competing microorganisms in the ecological niches they inhabit, using a wide range of various defense mechanisms including the production of a large number of secondary metabolic products, as well as many biologically active proteinaceous agents, such as bacteriocins. Strains that produce more than one bacteriocin with a narrow but different inhibitory spectrum have a better chance to survive in an environment shared with closely related species with which they are in competition for the same nutrients. A presence of huge number of bacteriocin producing strains could be explained by the fact that the bacteriocin genes were often located on mobile genetic elements such as conjugative plasmids or transposons, allowing their wide distribution via horizontal gene transfer. The aim of this doctoral dissertation was to determine the antimicrobial potential of 52 strains of Lactobacillus casei/paracasei group, followed by a comparative screening of the bacterial genomes in order to determine the distribution of BacSJ and acidocin 8912 encoding genes, and also to determine the correlation of presence of bacteriocin genes and bacteriocin production in the analyzed strains. This approach enabled identification of potentially new bacteriocins and putative bacteriocin genes regardless of whether the analyzed bacterial strains produced bacteriocin. Strains of Lb. casei/paracasei group analyzed in this study, isolates from fermented dairy products (different types of cheese and kajmak) produced in households, were determined through microbiological and biochemical tests and using molecular methods PCR reactions with (GTG)5 primer and sequencing of 16S rRNA gene. Detection of antimicrobial activity of strains from Lb. casei/paracasei group was performed by bacteriocin activity assay using sensitive Lactobacillus and Lactococcus indicator strains and cross-inhibition tests among bacteriocin producer strains. To determine the distribution of genes encoding bacteriocin BacSJ, acidocin 8912, ABCtransporter and accessory protein (Acc), PCR analysis with specific primers for those genes was used. For the re-establishment of the ability to produce BacSJ in BGNK1-62 strain, in which the presence of a potential operon bacSJ2-8/bacSJ2-8i was identified, plasmid pA2A was used. Plasmid pA2A was obtained by cloning of a PCR fragment carrying abcT and acc genes, involved in transport of bacteriocin BacSJ, in lactobacilli/lactococci/E. coli shuttle cloning vector pA13. Biochemical characterization of two new bacteriocins BacUB9 produced by strain Lb. paracasei subsp. paracasei BGUB9 and BacGR produced by strain Lb. paracasei subsp. paracasei BGGR2-66, was done by following the effects of proteolytic enzymes, different pH values and temperature on their activity. Isolation of bacteriocin BacUB9 from supernatant of the overnight culture was done in two ways: ammonium sulphate and acetone precipitation. The sample obtained by ammonium sulphate precipitation was purified by reversed-phase chromatography with a "step" gradient elution, and then analyzed on Tricin SDS-polyacrylamide gel. Sample obtained from acetone precipitation was used for determination of bacteriocin molecular mass by using mass spectrometry LC/MS. Isolation of bacteriocin BacGR, adsorbed on the surface of cells, was done with chloroform extraction after cultivation of BGGR2-66 strain on solid MRS medium for 48 hours. The sample obtained by chloroform extraction was then analyzed in two ways. The sample obtained from reverse-phase high performance liquid chromatography (HPLC) was used to determine the molecular weight of bacteriocin BacGR, by using mass spectrometry (ESI/MS). Also, a sample obtained from chloroform extraction was analyzed on discontinuous Tricin SDSpolyacrylamide gel, after which the sample was transferred to PVDF membrane and sent to N-terminal amino acid sequencing. The analysis of antimicrobial activity showed that 17 of 52 tested Lactobacillus casei/paracasei strains produced bacteriocins. Based on the similarity of the inhibitory spectrum, five groups of strains we defined (A-E). Group A includes strains that have a similar antimicrobial spectrum as previously characterized bacteriocin producer strain BGSJ2-8, and group C includes strains that have a similar spectrum as characterized bacteriocin producer strain BGBUK2-16. Because the use of classical methods for detection of bacteriocin producers may underestimate the bacteriocinogenic potential of tested strains, all 52 strains of Lb. casei/paracasei group, not only the strains with a similar antimicrobial spectrum as strain BGSJ2-8, were screened by PCR amplification for presence of relevant bacteriocin genes. It was shown that the genetic determinants required for the production of bacteriocins BacSJ (bacSJ2-8/bacSJ2-8i operon) and/or acidocin 8912 (acdT gene) were widespread within the Lb. casei/paracasei group with a frequency of 96.15%. On the basis of whether they produce bacteriocins, and whether they have or do not possess the analyzed bacteriocin genes, strains of Lb. casei/paracasei group were divided into two main groups: bacteriocin producer strains (P1-P7) and strains in which the bacteriocin activity was not detected (N1-N8). In all bacteriocin producers the presence of both or at least one structural bacteriocin gene (bacSJ2-8/bacSJ2-8i operon and/or acdT gene) was identified. A common feature of strains from P3 group, which distinguishes them from strains from P1 group, is the absence of abcT gene. The narrow spectrum of activity of three of the five strains of P3 group (BGGR2-64, BGGR2-66 and BGBUK2-16) can be explained by the inability to compensate for the absence of abcT gene, as well as with production of potentially new bacteriocin. The common characteristic of strains belonging to groups P2 and P4-P7 is a lack of acc gene, which narrow spectrum of activity can be explained by the loss of acc gene, which causes their poor bacteriocin activity, or by production of new bacteriocin, which activity does not require the presence of accessory protein. Interestingly, in the genomes of 33 strains of Lb. casei/paracasei group, that do not produce bacteriocins, the presence of one or both putative structural genes for production of bacteriocin BacSJ and acidocin 8912 were identified. Absence of ability to produce bacteriocins can be explained by the presence of mutations in the genes or regulatory sequences of genes encoding components involved in the synthesis and secretion of bacteriocins. It is also possible that the structural bacteriocin genes were expressed, but that the loss abcT gene (N3 and N6) and the lack of acc gene (N2, N4, N5 and N7) caused the absence of secretion of the synthesized bacteriocin. Re-establishment of the ability of strain BGNK1-62 to produce bacteriocin by in trans expression of heterologous abcT and acc genes illustrated the importance of identifying putative bacteriocin genes in the analysis of antimicrobial potential of bacterial strains of interest. This study showed that both strains BGUB9 and BGGR2-66 possess genetic potential to produce three bacteriocins, but that their antimicrobial activities probably were result of production of new, until now not characterized bacteriocins BacUB9 and BacGR. DNA-DNA hybridization showed plasmid localization of putative genes involved in production of bacteriocin BacSJ and acidocin 8912 in genome of the BGUB9 and BGGR2-66 strains, and also Bac-derivatives of these strains, obtained after plasmid curing experiments, indicated that the genetic determinants necessary for production of bacteriocins BacUB9 and BacGR were plasmid-encoded. BacUB9 is a relatively heat-stable, low-molecular-weight peptide (3758 Da), active within a broad pH range, with a bacteriostatic mode of action, sensitive to proteolytic enzymes, which beside closely related species also inhibits the growth of Listeria innocua ATCC 33090T strain. Based on these characteristics it appears that BacUB9 belongs to the class IIa bacteriocins. Bacteriocin BacGR is also small (3218 Da) and relatively heat-stable peptide, active in a wide range of pH values, inactivated by proteolytic enzymes, but with a narrow spectrum of activity limited to closely related species. Based on the molecular size, biochemical characterization and obtained N-terminal sequence, bacteriocin BacGR probably belongs to the class IId bacteriocins.- Veliki broj vrsta roda Lactobacillus zauzima ključno mesto u proizvodnji fermentisanih mlečnih i mesnih proizvoda, prisutni su i u fermentisanom povrću i proizvodima od žitarica. Laktobacili se takođe koriste i u medicini usled pozitivnih efekata koje pojedini sojevi imaju na zdravlje domaćina. Pojedini sojevi laktobacila imaju i probiotski potencijal zahvaljujući sposobnosti produkcije različitih antimikrobnih jedinjenja, egzopolisaharida (EPS), proteinaza i dr. Vrste roda Lactobacillus poseduju potencijal da inhibiraju rast konkurentskih mikroorganizama u ekološkim nišama koje naseljavaju, korišćenjem širokog spektra različitih odbrambenih mehanizama uključujući proizvodnju velikog broja metaboličkih nusproizvoda, kao i mnogih biološki aktivnih agenasa proteinske prirode kao što bakteriocini. Sojevi koji proizvode više od jednog bakteriocina sa uskim, ali različitim inhibitornim spektrom, imaju veće šanse za opstanak u okruženj