TY  - JOUR
AU  - Malešević, Milka
AU  - Gardijan, Lazar
AU  - Miljković, Marija
AU  - O'Connor, Paula M
AU  - Mirković, Nemanja
AU  - Jovčić, Branko
AU  - Cotter, Paul D
AU  - Jovanovic, Goran
AU  - Kojić, Milan
PY  - 2023
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/1828
UR  - http://intor.torlakinstitut.com/handle/123456789/740
AB  - Lactic acid bacterium Lactococcus lactis BGBU1-4 produces 43 amino acids (aa) long bacteriocin, lactolisterin BU (LBU), a 5.161 kDa peptide with potent antibacterial activity against many Gram-positive pathogens. In addition, BGBU1-4 produces an additional unknown product of 3.642 kDa with antibacterial activity. Here, we determined that the significant amount of naturally produced LBU breaks down to create a 3.642 kDa truncated form of LBU bacteriocin consisting of 31 N-terminal aa (LBU1-31) that exhibits 12.5% the antibacterial activity of the full-length LBU. We showed that chemically synthesized LBU is stable and 50% less active than native LBU, and so we used the synthetic peptides of LBU and its variants to further study their activities and antibacterial potential. Deletion analysis of LBU revealed that the 24 N-terminal aa of LBU (LBU1-24) are responsible for antibacterial activity, while downstream aa (25–43) determine the species-specific effectiveness of LBU. Although LBU1-31 contains aa 1–24, the truncation at position 31 is predicted to change the structure within aa 15–31 and might impact on antibacterial activity. Intriguingly, whole genome sequencing and genome mining established that BGBU1-4 is abundant in genes that encode potential antibacterials, but produces LBU and its breakdown product LBU1-31 exclusively.
T2  - Letters in Applied Microbiology
T2  - Letters in Applied MicrobiologyLetters in Applied Microbiology
T1  - Exploring the antibacterial potential of Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 by genome mining, bacteriocin gene overexpression, and chemical protein synthesis of lactolisterin BU variants
IS  - 2
SP  - ovad004
VL  - 76
DO  - 10.1093/lambio/ovad004
ER  - 

@article{
author = "Malešević, Milka and Gardijan, Lazar and Miljković, Marija and O'Connor, Paula M and Mirković, Nemanja and Jovčić, Branko and Cotter, Paul D and Jovanovic, Goran and Kojić, Milan",
year = "2023",
abstract = "Lactic acid bacterium Lactococcus lactis BGBU1-4 produces 43 amino acids (aa) long bacteriocin, lactolisterin BU (LBU), a 5.161 kDa peptide with potent antibacterial activity against many Gram-positive pathogens. In addition, BGBU1-4 produces an additional unknown product of 3.642 kDa with antibacterial activity. Here, we determined that the significant amount of naturally produced LBU breaks down to create a 3.642 kDa truncated form of LBU bacteriocin consisting of 31 N-terminal aa (LBU1-31) that exhibits 12.5% the antibacterial activity of the full-length LBU. We showed that chemically synthesized LBU is stable and 50% less active than native LBU, and so we used the synthetic peptides of LBU and its variants to further study their activities and antibacterial potential. Deletion analysis of LBU revealed that the 24 N-terminal aa of LBU (LBU1-24) are responsible for antibacterial activity, while downstream aa (25–43) determine the species-specific effectiveness of LBU. Although LBU1-31 contains aa 1–24, the truncation at position 31 is predicted to change the structure within aa 15–31 and might impact on antibacterial activity. Intriguingly, whole genome sequencing and genome mining established that BGBU1-4 is abundant in genes that encode potential antibacterials, but produces LBU and its breakdown product LBU1-31 exclusively.",
journal = "Letters in Applied Microbiology, Letters in Applied MicrobiologyLetters in Applied Microbiology",
title = "Exploring the antibacterial potential of Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 by genome mining, bacteriocin gene overexpression, and chemical protein synthesis of lactolisterin BU variants",
number = "2",
pages = "ovad004",
volume = "76",
doi = "10.1093/lambio/ovad004"
}

Malešević, M., Gardijan, L., Miljković, M., O'Connor, P. M., Mirković, N., Jovčić, B., Cotter, P. D., Jovanovic, G.,& Kojić, M.. (2023). Exploring the antibacterial potential of Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 by genome mining, bacteriocin gene overexpression, and chemical protein synthesis of lactolisterin BU variants. in Letters in Applied Microbiology, 76(2), ovad004.
https://doi.org/10.1093/lambio/ovad004

Malešević M, Gardijan L, Miljković M, O'Connor PM, Mirković N, Jovčić B, Cotter PD, Jovanovic G, Kojić M. Exploring the antibacterial potential of Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 by genome mining, bacteriocin gene overexpression, and chemical protein synthesis of lactolisterin BU variants. in Letters in Applied Microbiology. 2023;76(2):ovad004.
doi:10.1093/lambio/ovad004 .

Malešević, Milka, Gardijan, Lazar, Miljković, Marija, O'Connor, Paula M, Mirković, Nemanja, Jovčić, Branko, Cotter, Paul D, Jovanovic, Goran, Kojić, Milan, "Exploring the antibacterial potential of Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 by genome mining, bacteriocin gene overexpression, and chemical protein synthesis of lactolisterin BU variants" in Letters in Applied Microbiology, 76, no. 2 (2023):ovad004,
https://doi.org/10.1093/lambio/ovad004 . .

TY  - JOUR
AU  - Mirković, Nemanja
AU  - Obradović, Mina
AU  - O'Connor, Paula M.
AU  - Filipić, Brankica
AU  - Jovčić, Branko
AU  - Cotter, Paul D.
AU  - Kojić, Milan
PY  - 2021
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/1429
UR  - http://intor.torlakinstitut.com/handle/123456789/706
AB  - Screening for producers of potent antimicrobial peptides, resulted in the isolation of Bacillus cereus BGNM1 with strong antimicrobial activity against Listeria monocytogenes. Genome sequence analysis revealed that BGNM1 contains the gene cluster associated with the production of the lantibiotic, thusin, previously identified in B. thuringiensis. Purification of the antimicrobial activity confirmed that strain BGMN1 produces thusin. Both thusin sensitive and resistant strains were detected among clinical isolates of Streptococcus agalactiae. Random mutagenesis of a thusin sensitive strain, S. agalactiae B782, was performed in an attempt to identify the receptor protein for thusin. Three independent thusin resistant mutants were selected and their complete genomes sequenced. Comparative sequence analysis of these mutants with the WT strain revealed that duplication of a region encoding a 79 amino acids repeat in a C-protein alpha-antigen was a common difference, suggesting it to be responsible for increased resistance to thusin. Since induced thusin resistant mutants showed higher level of resistance than the naturally resistant B761 strain, complete genome sequencing of strain B761 was performed to check the integrity of the C-protein alpha-antigen-encoding gene. This analysis revealed that this gene is deleted in B761, providing further evidence that this protein promotes interaction of the thusin with receptor.
PB  - Springer, Dordrecht
T2  - Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology
T1  - C-protein alpha-antigen modulates the lantibiotic thusin resistance in Streptococcus agalactiae
EP  - 1607
IS  - 10
SP  - 1595
VL  - 114
DO  - 10.1007/s10482-021-01626-3
ER  - 

@article{
author = "Mirković, Nemanja and Obradović, Mina and O'Connor, Paula M. and Filipić, Brankica and Jovčić, Branko and Cotter, Paul D. and Kojić, Milan",
year = "2021",
abstract = "Screening for producers of potent antimicrobial peptides, resulted in the isolation of Bacillus cereus BGNM1 with strong antimicrobial activity against Listeria monocytogenes. Genome sequence analysis revealed that BGNM1 contains the gene cluster associated with the production of the lantibiotic, thusin, previously identified in B. thuringiensis. Purification of the antimicrobial activity confirmed that strain BGMN1 produces thusin. Both thusin sensitive and resistant strains were detected among clinical isolates of Streptococcus agalactiae. Random mutagenesis of a thusin sensitive strain, S. agalactiae B782, was performed in an attempt to identify the receptor protein for thusin. Three independent thusin resistant mutants were selected and their complete genomes sequenced. Comparative sequence analysis of these mutants with the WT strain revealed that duplication of a region encoding a 79 amino acids repeat in a C-protein alpha-antigen was a common difference, suggesting it to be responsible for increased resistance to thusin. Since induced thusin resistant mutants showed higher level of resistance than the naturally resistant B761 strain, complete genome sequencing of strain B761 was performed to check the integrity of the C-protein alpha-antigen-encoding gene. This analysis revealed that this gene is deleted in B761, providing further evidence that this protein promotes interaction of the thusin with receptor.",
publisher = "Springer, Dordrecht",
journal = "Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology",
title = "C-protein alpha-antigen modulates the lantibiotic thusin resistance in Streptococcus agalactiae",
pages = "1607-1595",
number = "10",
volume = "114",
doi = "10.1007/s10482-021-01626-3"
}

Mirković, N., Obradović, M., O'Connor, P. M., Filipić, B., Jovčić, B., Cotter, P. D.,& Kojić, M.. (2021). C-protein alpha-antigen modulates the lantibiotic thusin resistance in Streptococcus agalactiae. in Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology
Springer, Dordrecht., 114(10), 1595-1607.
https://doi.org/10.1007/s10482-021-01626-3

Mirković N, Obradović M, O'Connor PM, Filipić B, Jovčić B, Cotter PD, Kojić M. C-protein alpha-antigen modulates the lantibiotic thusin resistance in Streptococcus agalactiae. in Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology. 2021;114(10):1595-1607.
doi:10.1007/s10482-021-01626-3 .

Mirković, Nemanja, Obradović, Mina, O'Connor, Paula M., Filipić, Brankica, Jovčić, Branko, Cotter, Paul D., Kojić, Milan, "C-protein alpha-antigen modulates the lantibiotic thusin resistance in Streptococcus agalactiae" in Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, 114, no. 10 (2021):1595-1607,
https://doi.org/10.1007/s10482-021-01626-3 . .

TY  - JOUR
AU  - Miljković, Marija
AU  - Jovanović, Sofija
AU  - O'Connor, Paula M.
AU  - Mirković, Nemanja
AU  - Jovčić, Branko
AU  - Filipić, Brankica
AU  - Dinić, Miroslav
AU  - Studholme, David John
AU  - Fira, Đorđe
AU  - Cotter, Paul D.
AU  - Kojić, Milan
PY  - 2019
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/1264
UR  - http://intor.torlakinstitut.com/handle/123456789/702
AB  - Bacteria active against multi-drug resistant pathogens, isolated by direct selection of colonies from clover silage samples, produce zones of inhibition against two Gram-negative (Klebsiella pneumoniae Ni9 and Pseudomonas aeruginosa MMA83) and two Gram-positive (Staphylococcus aureus ATCC25923 and Listeria monocytogenes ATCC19111) pathogens. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 produced the largest zones of inhibition against all four pathogens when grown in LB broth with aeration at 37 degrees C. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 were identified as Brevibacillus laterosporus and pulsed field gel electrophoresis and extracellular protein profiles showed that three different strains (BGSP7, BGSP9 and BGSP11) were isolated. A semi-native SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) gel overlay assay showed that BGSP7 and BGSP9 produce small antimicrobial molecules of about 1.5 kDa, while BGSP11 produces antimicrobial molecules of 1.5 and 6 kDa active against S. aureus ATCC25923. Amino acid analysis of two antimicrobial molecules (1583.73 Da; from BGSP7 and 1556.31 Da; from BGSP11) revealed that they have a similar composition and differ only by virtue of the presence of a methionine which is present only in BGSP11 molecule. Genome sequencing of the three isolates revealed the presence of gene clusters associated with the production of non-ribosomally synthesized peptides (brevibacillin, bogorol, gramicidin S, plipastatin and tyrocin) and bacteriocins (laterosporulin, a lactococcin 972-like bacteriocin, as well as putative linocin M18, sactipeptide, UviB and lantipeptide-like molecules). Ultimately, the purification of a number of antimicrobial molecules from each isolate suggests that they can be considered as potent biocontrol strains that produce an arsenal of antimicrobial molecules active against Gram-positive and Gram-negative multi-resistant pathogens, fungi and insects.
PB  - Public Library Science, San Francisco
T2  - PLoS One
T1  - Brevibacillus laterosporus strains BGSP7, BGSP9 and BGSP11 isolated from silage produce broad spectrum multi-antimicrobials
IS  - 5
VL  - 14
DO  - 10.1371/journal.pone.0216773
ER  - 

@article{
author = "Miljković, Marija and Jovanović, Sofija and O'Connor, Paula M. and Mirković, Nemanja and Jovčić, Branko and Filipić, Brankica and Dinić, Miroslav and Studholme, David John and Fira, Đorđe and Cotter, Paul D. and Kojić, Milan",
year = "2019",
abstract = "Bacteria active against multi-drug resistant pathogens, isolated by direct selection of colonies from clover silage samples, produce zones of inhibition against two Gram-negative (Klebsiella pneumoniae Ni9 and Pseudomonas aeruginosa MMA83) and two Gram-positive (Staphylococcus aureus ATCC25923 and Listeria monocytogenes ATCC19111) pathogens. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 produced the largest zones of inhibition against all four pathogens when grown in LB broth with aeration at 37 degrees C. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 were identified as Brevibacillus laterosporus and pulsed field gel electrophoresis and extracellular protein profiles showed that three different strains (BGSP7, BGSP9 and BGSP11) were isolated. A semi-native SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) gel overlay assay showed that BGSP7 and BGSP9 produce small antimicrobial molecules of about 1.5 kDa, while BGSP11 produces antimicrobial molecules of 1.5 and 6 kDa active against S. aureus ATCC25923. Amino acid analysis of two antimicrobial molecules (1583.73 Da; from BGSP7 and 1556.31 Da; from BGSP11) revealed that they have a similar composition and differ only by virtue of the presence of a methionine which is present only in BGSP11 molecule. Genome sequencing of the three isolates revealed the presence of gene clusters associated with the production of non-ribosomally synthesized peptides (brevibacillin, bogorol, gramicidin S, plipastatin and tyrocin) and bacteriocins (laterosporulin, a lactococcin 972-like bacteriocin, as well as putative linocin M18, sactipeptide, UviB and lantipeptide-like molecules). Ultimately, the purification of a number of antimicrobial molecules from each isolate suggests that they can be considered as potent biocontrol strains that produce an arsenal of antimicrobial molecules active against Gram-positive and Gram-negative multi-resistant pathogens, fungi and insects.",
publisher = "Public Library Science, San Francisco",
journal = "PLoS One",
title = "Brevibacillus laterosporus strains BGSP7, BGSP9 and BGSP11 isolated from silage produce broad spectrum multi-antimicrobials",
number = "5",
volume = "14",
doi = "10.1371/journal.pone.0216773"
}

Miljković, M., Jovanović, S., O'Connor, P. M., Mirković, N., Jovčić, B., Filipić, B., Dinić, M., Studholme, D. J., Fira, Đ., Cotter, P. D.,& Kojić, M.. (2019). Brevibacillus laterosporus strains BGSP7, BGSP9 and BGSP11 isolated from silage produce broad spectrum multi-antimicrobials. in PLoS One
Public Library Science, San Francisco., 14(5).
https://doi.org/10.1371/journal.pone.0216773

Miljković M, Jovanović S, O'Connor PM, Mirković N, Jovčić B, Filipić B, Dinić M, Studholme DJ, Fira Đ, Cotter PD, Kojić M. Brevibacillus laterosporus strains BGSP7, BGSP9 and BGSP11 isolated from silage produce broad spectrum multi-antimicrobials. in PLoS One. 2019;14(5).
doi:10.1371/journal.pone.0216773 .

Miljković, Marija, Jovanović, Sofija, O'Connor, Paula M., Mirković, Nemanja, Jovčić, Branko, Filipić, Brankica, Dinić, Miroslav, Studholme, David John, Fira, Đorđe, Cotter, Paul D., Kojić, Milan, "Brevibacillus laterosporus strains BGSP7, BGSP9 and BGSP11 isolated from silage produce broad spectrum multi-antimicrobials" in PLoS One, 14, no. 5 (2019),
https://doi.org/10.1371/journal.pone.0216773 . .

TY  - JOUR
AU  - Miljković, Manja
AU  - Lozo, Jelena
AU  - Mirković, Nemanja
AU  - O'Connor, Paula M.
AU  - Malešević, Milka
AU  - Jovčić, Branko
AU  - Cotter, Paul D.
AU  - Kojić, Milan
PY  - 2018
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/1124
UR  - http://intor.torlakinstitut.com/handle/123456789/743
AB  - The gene cluster responsible for the production of the aureocin A53-like bacteriocin, lactolisterin BU, is located on plasmid pBU6 in Lactococcus lactis subsp. lactis BGBU1-4. Heterologous expression of pBU6 confirmed that production and limited immunity to lactolisterin BU were provided by the plasmid. Comparative analysis of aureocin A53-like operons revealed that the structural genes shared a low level of identity, while other genes were without homology, indicating a different origin. Subcloning and expression of genes located downstream of the structural gene, IliBU, revealed that the lactolisterin BU cluster consists of four genes: the structural gene IliBU, the abcT gene encoding an ABC transporter, the accL gene encoding an accessory protein and the immL gene which provides limited immunity to lactolisterin BU. Reverse transcription analysis revealed that all genes were transcribed as one polycistronic mRNA. Attempts to split the lactolisterin BU operon, even when both parts were under control of the PlliBU promoter, were unsuccessful indicating that expression of lactolisterin BU is probably precisely regulated at the translational level by translational coupling and is possible only when all genes of the operon are in cis constellation. Two rho-independent transcription terminators were detected in the lactolisterin BU operon: the first in the intergenic region of the IliBU and abcT genes and the second at the end of operon. Deletion of the second transcription terminator did not influence production of the bacteriocin in lactococci.
PB  - Frontiers Media Sa, Lausanne
T2  - Frontiers in Microbiology
T1  - Functional Characterization of the Lactolisterin BU Gene Cluster of Lactococcus lactis subsp. lactis BGBU1-4
VL  - 9
DO  - 10.3389/fmicb.2018.02774
ER  - 

@article{
author = "Miljković, Manja and Lozo, Jelena and Mirković, Nemanja and O'Connor, Paula M. and Malešević, Milka and Jovčić, Branko and Cotter, Paul D. and Kojić, Milan",
year = "2018",
abstract = "The gene cluster responsible for the production of the aureocin A53-like bacteriocin, lactolisterin BU, is located on plasmid pBU6 in Lactococcus lactis subsp. lactis BGBU1-4. Heterologous expression of pBU6 confirmed that production and limited immunity to lactolisterin BU were provided by the plasmid. Comparative analysis of aureocin A53-like operons revealed that the structural genes shared a low level of identity, while other genes were without homology, indicating a different origin. Subcloning and expression of genes located downstream of the structural gene, IliBU, revealed that the lactolisterin BU cluster consists of four genes: the structural gene IliBU, the abcT gene encoding an ABC transporter, the accL gene encoding an accessory protein and the immL gene which provides limited immunity to lactolisterin BU. Reverse transcription analysis revealed that all genes were transcribed as one polycistronic mRNA. Attempts to split the lactolisterin BU operon, even when both parts were under control of the PlliBU promoter, were unsuccessful indicating that expression of lactolisterin BU is probably precisely regulated at the translational level by translational coupling and is possible only when all genes of the operon are in cis constellation. Two rho-independent transcription terminators were detected in the lactolisterin BU operon: the first in the intergenic region of the IliBU and abcT genes and the second at the end of operon. Deletion of the second transcription terminator did not influence production of the bacteriocin in lactococci.",
publisher = "Frontiers Media Sa, Lausanne",
journal = "Frontiers in Microbiology",
title = "Functional Characterization of the Lactolisterin BU Gene Cluster of Lactococcus lactis subsp. lactis BGBU1-4",
volume = "9",
doi = "10.3389/fmicb.2018.02774"
}

Miljković, M., Lozo, J., Mirković, N., O'Connor, P. M., Malešević, M., Jovčić, B., Cotter, P. D.,& Kojić, M.. (2018). Functional Characterization of the Lactolisterin BU Gene Cluster of Lactococcus lactis subsp. lactis BGBU1-4. in Frontiers in Microbiology
Frontiers Media Sa, Lausanne., 9.
https://doi.org/10.3389/fmicb.2018.02774

Miljković M, Lozo J, Mirković N, O'Connor PM, Malešević M, Jovčić B, Cotter PD, Kojić M. Functional Characterization of the Lactolisterin BU Gene Cluster of Lactococcus lactis subsp. lactis BGBU1-4. in Frontiers in Microbiology. 2018;9.
doi:10.3389/fmicb.2018.02774 .

Miljković, Manja, Lozo, Jelena, Mirković, Nemanja, O'Connor, Paula M., Malešević, Milka, Jovčić, Branko, Cotter, Paul D., Kojić, Milan, "Functional Characterization of the Lactolisterin BU Gene Cluster of Lactococcus lactis subsp. lactis BGBU1-4" in Frontiers in Microbiology, 9 (2018),
https://doi.org/10.3389/fmicb.2018.02774 . .