TY  - JOUR
AU  - Mirković, Nemanja
AU  - Polović, Natalija
AU  - Vukotić, Goran
AU  - Jovčić, Branko
AU  - Miljković, Marija
AU  - Radulović, Zorica
AU  - Diep, Dzung B.
AU  - Kojić, Milan
PY  - 2016
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/980
AB  - Bacteriocin producers normally possess dedicated immunity systems to protect themselves from their own bacteriocins. Lactococcus lactis strains LMG2081 and BGBM50 are known as lactococcin G producers. However, BGBM50 was sensitive to LMG2081, which indicated that LMG2081 might produce additional bacteriocins that are not present in BGBM50. Therefore, whole-genome sequencing of the two strains was performed, and a lantibiotic operon (called lctLMG) was identified in LMG2081 but not in BGBM50. The lctLMG operon contains six open reading frames; the first three genes, lmgA, lmgM, and lmgT, are involved in the biosynthesis and export of bacteriocin, while the other three genes, lmgF, lmgE, and lmgG, are involved in lantibiotic immunity. Mutational analysis confirmed that the lctLMG operon is responsible for the additional antimicrobial activity. Specifically, site-directed mutation within this operon rendered LMG2081 inactive toward BGBM50. Subsequent purification and electrospray ionization-time of flight mass spectrometric analysis confirmed that the lantibiotic bacteriocin called lacticin LMG is exported as a 25-amino-acid peptide. Lacticin LMG is highly similar to the lacticin 481 group. It is interesting that a bacteriocin producer produces two different classes of bacteriocins, whose operons are located in the chromosome and a plasmid.
PB  - Amer Soc Microbiology, Washington
T2  - Applied and Environmental Microbiology
T1  - Lactococcus lactis LMG2081 Produces Two Bacteriocins, a Nonlantibiotic and a Novel Lantibiotic
EP  - 2562
IS  - 8
SP  - 2555
VL  - 82
DO  - 10.1128/AEM.03988-15
ER  - 

@article{
author = "Mirković, Nemanja and Polović, Natalija and Vukotić, Goran and Jovčić, Branko and Miljković, Marija and Radulović, Zorica and Diep, Dzung B. and Kojić, Milan",
year = "2016",
abstract = "Bacteriocin producers normally possess dedicated immunity systems to protect themselves from their own bacteriocins. Lactococcus lactis strains LMG2081 and BGBM50 are known as lactococcin G producers. However, BGBM50 was sensitive to LMG2081, which indicated that LMG2081 might produce additional bacteriocins that are not present in BGBM50. Therefore, whole-genome sequencing of the two strains was performed, and a lantibiotic operon (called lctLMG) was identified in LMG2081 but not in BGBM50. The lctLMG operon contains six open reading frames; the first three genes, lmgA, lmgM, and lmgT, are involved in the biosynthesis and export of bacteriocin, while the other three genes, lmgF, lmgE, and lmgG, are involved in lantibiotic immunity. Mutational analysis confirmed that the lctLMG operon is responsible for the additional antimicrobial activity. Specifically, site-directed mutation within this operon rendered LMG2081 inactive toward BGBM50. Subsequent purification and electrospray ionization-time of flight mass spectrometric analysis confirmed that the lantibiotic bacteriocin called lacticin LMG is exported as a 25-amino-acid peptide. Lacticin LMG is highly similar to the lacticin 481 group. It is interesting that a bacteriocin producer produces two different classes of bacteriocins, whose operons are located in the chromosome and a plasmid.",
publisher = "Amer Soc Microbiology, Washington",
journal = "Applied and Environmental Microbiology",
title = "Lactococcus lactis LMG2081 Produces Two Bacteriocins, a Nonlantibiotic and a Novel Lantibiotic",
pages = "2562-2555",
number = "8",
volume = "82",
doi = "10.1128/AEM.03988-15"
}

Mirković, N., Polović, N., Vukotić, G., Jovčić, B., Miljković, M., Radulović, Z., Diep, D. B.,& Kojić, M.. (2016). Lactococcus lactis LMG2081 Produces Two Bacteriocins, a Nonlantibiotic and a Novel Lantibiotic. in Applied and Environmental Microbiology
Amer Soc Microbiology, Washington., 82(8), 2555-2562.
https://doi.org/10.1128/AEM.03988-15

Mirković N, Polović N, Vukotić G, Jovčić B, Miljković M, Radulović Z, Diep DB, Kojić M. Lactococcus lactis LMG2081 Produces Two Bacteriocins, a Nonlantibiotic and a Novel Lantibiotic. in Applied and Environmental Microbiology. 2016;82(8):2555-2562.
doi:10.1128/AEM.03988-15 .

Mirković, Nemanja, Polović, Natalija, Vukotić, Goran, Jovčić, Branko, Miljković, Marija, Radulović, Zorica, Diep, Dzung B., Kojić, Milan, "Lactococcus lactis LMG2081 Produces Two Bacteriocins, a Nonlantibiotic and a Novel Lantibiotic" in Applied and Environmental Microbiology, 82, no. 8 (2016):2555-2562,
https://doi.org/10.1128/AEM.03988-15 . .

TY  - JOUR
AU  - Miljković, Marija
AU  - Uzelac, Gordana
AU  - Mirković, Nemanja
AU  - Devescovi, Giulia
AU  - Diep, Dzung B.
AU  - Venturi, Vittorio
AU  - Kojić, Milan
PY  - 2016
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/904
AB  - The Zn-dependent membrane-located protease YvjB has previously been shown to serve as a target receptor for LsbB, a class II leaderless lactococcal bacteriocin. Although yvjB is highly conserved in the genus Lactococcus, the bacteriocin appears to be active only against the subspecies L. lactis subsp. lactis. Comparative analysis of the YvjB proteins of a sensitive strain (YvjB(MN)) and a resistant strain (YvjB(MG)) showed that they differ from each other in 31 positions. In this study, we applied site-directed mutagenesis and performed directed binding studies to provide biochemical evidence that LsbB interacts with the third transmembrane helix of YvjB in susceptible cells. The site-directed mutagenesis of LsbB and YvjB proteins showed that certain amino acids and the length of LsbB are responsible for the bacteriocin activity, most probably through adequate interaction of these two proteins; the essential amino acids in LsbB responsible for the activity are tryptophan (Trp(25)) and terminal alanine (Ala(30)). It was also shown that the distance between Trp(25) and terminal alanine is crucial for LsbB activity. The crucial region in YvjB for the interaction with LsbB is the beginning of the third transmembrane helix, particularly amino acids tyrosine (Tyr(356)) and alanine (Ala(353)). In vitro experiments showed that LsbB could interact with both YvjB(MN) and YvjB(MG), but the strength of interaction is significantly less with YvjB(MG). In vivo experiments with immunofluorescently labeled antibody demonstrated that LsbB specifically interacts only with cells carrying YvjB(MN). IMPORTANCE The antimicrobial activity of LsbB bacteriocin depends on the correct interaction with the corresponding receptor in the bacterial membrane of sensitive cells. Membrane-located bacteriocin receptors have essential primary functions, such as cell wall synthesis or sugar transport, and it seems that interaction with bacteriocins is suicidal for cells. This study showed that the C-terminal part of LsbB is crucial for the bacteriocin activity, most probably through adequate interaction with the third transmembrane domain of the YvjB receptor. The conserved Tyr(356) and Ala(353) residues of YvjB are essential for the function of this Zn-dependent membrane-located protease as a bacteriocin receptor.
PB  - Amer Soc Microbiology, Washington
T2  - Applied and Environmental Microbiology
T1  - LsbB Bacteriocin Interacts with the Third Transmembrane Domain of the YvjB Receptor
EP  - 5374
IS  - 17
SP  - 5364
VL  - 82
DO  - 10.1128/AEM.01293-16
ER  - 

@article{
author = "Miljković, Marija and Uzelac, Gordana and Mirković, Nemanja and Devescovi, Giulia and Diep, Dzung B. and Venturi, Vittorio and Kojić, Milan",
year = "2016",
abstract = "The Zn-dependent membrane-located protease YvjB has previously been shown to serve as a target receptor for LsbB, a class II leaderless lactococcal bacteriocin. Although yvjB is highly conserved in the genus Lactococcus, the bacteriocin appears to be active only against the subspecies L. lactis subsp. lactis. Comparative analysis of the YvjB proteins of a sensitive strain (YvjB(MN)) and a resistant strain (YvjB(MG)) showed that they differ from each other in 31 positions. In this study, we applied site-directed mutagenesis and performed directed binding studies to provide biochemical evidence that LsbB interacts with the third transmembrane helix of YvjB in susceptible cells. The site-directed mutagenesis of LsbB and YvjB proteins showed that certain amino acids and the length of LsbB are responsible for the bacteriocin activity, most probably through adequate interaction of these two proteins; the essential amino acids in LsbB responsible for the activity are tryptophan (Trp(25)) and terminal alanine (Ala(30)). It was also shown that the distance between Trp(25) and terminal alanine is crucial for LsbB activity. The crucial region in YvjB for the interaction with LsbB is the beginning of the third transmembrane helix, particularly amino acids tyrosine (Tyr(356)) and alanine (Ala(353)). In vitro experiments showed that LsbB could interact with both YvjB(MN) and YvjB(MG), but the strength of interaction is significantly less with YvjB(MG). In vivo experiments with immunofluorescently labeled antibody demonstrated that LsbB specifically interacts only with cells carrying YvjB(MN). IMPORTANCE The antimicrobial activity of LsbB bacteriocin depends on the correct interaction with the corresponding receptor in the bacterial membrane of sensitive cells. Membrane-located bacteriocin receptors have essential primary functions, such as cell wall synthesis or sugar transport, and it seems that interaction with bacteriocins is suicidal for cells. This study showed that the C-terminal part of LsbB is crucial for the bacteriocin activity, most probably through adequate interaction with the third transmembrane domain of the YvjB receptor. The conserved Tyr(356) and Ala(353) residues of YvjB are essential for the function of this Zn-dependent membrane-located protease as a bacteriocin receptor.",
publisher = "Amer Soc Microbiology, Washington",
journal = "Applied and Environmental Microbiology",
title = "LsbB Bacteriocin Interacts with the Third Transmembrane Domain of the YvjB Receptor",
pages = "5374-5364",
number = "17",
volume = "82",
doi = "10.1128/AEM.01293-16"
}

Miljković, M., Uzelac, G., Mirković, N., Devescovi, G., Diep, D. B., Venturi, V.,& Kojić, M.. (2016). LsbB Bacteriocin Interacts with the Third Transmembrane Domain of the YvjB Receptor. in Applied and Environmental Microbiology
Amer Soc Microbiology, Washington., 82(17), 5364-5374.
https://doi.org/10.1128/AEM.01293-16

Miljković M, Uzelac G, Mirković N, Devescovi G, Diep DB, Venturi V, Kojić M. LsbB Bacteriocin Interacts with the Third Transmembrane Domain of the YvjB Receptor. in Applied and Environmental Microbiology. 2016;82(17):5364-5374.
doi:10.1128/AEM.01293-16 .

Miljković, Marija, Uzelac, Gordana, Mirković, Nemanja, Devescovi, Giulia, Diep, Dzung B., Venturi, Vittorio, Kojić, Milan, "LsbB Bacteriocin Interacts with the Third Transmembrane Domain of the YvjB Receptor" in Applied and Environmental Microbiology, 82, no. 17 (2016):5364-5374,
https://doi.org/10.1128/AEM.01293-16 . .

TY  - JOUR
AU  - Ovchinnikov, Kirill V.
AU  - Kristiansen, Per E.
AU  - Uzelac, Gordana
AU  - Topisirović, Ljubiša
AU  - Kojić, Milan
AU  - Nissen-Meyer, Jon
AU  - Nes, Ingolf F.
AU  - Diep, Dzung B.
PY  - 2014
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/750
AB  - Background: The bacteriocin LsbB targets a membrane-bound zinc-dependent peptidase. Results: The structure of LsbB was resolved by NMR. The C-terminal unstructured domains of LsbB and several other related bacteriocins were responsible for receptor binding. Conclusion: A subgroup of leaderless bacteriocins has been found to share a similar mechanism in receptor recognition. Significance: The study highlights the structure-function relationship of LsbB. LsbB is a class II leaderless lactococcal bacteriocin of 30 amino acids. In the present work, the structure and function relationship of LsbB was assessed. Structure determination by NMR spectroscopy showed that LsbB has an N-terminal -helix, whereas the C-terminal of the molecule remains unstructured. To define the receptor binding domain of LsbB, a competition assay was performed in which a systematic collection of truncated peptides of various lengths covering different parts of LsbB was used to inhibit the antimicrobial activity of LsbB. The results indicate that the outmost eight-amino acid sequence at the C-terminal end is likely to contain the receptor binding domain because only truncated fragments from this region could antagonize the antimicrobial activity of LsbB. Furthermore, alanine substitution revealed that the tryptophan in position 25 (Trp(25)) is crucial for the blocking activity of the truncated peptides, as well as for the antimicrobial activity of the full-length bacteriocin. LsbB shares significant sequence homology with five other leaderless bacteriocins, especially at their C-terminal halves where all contain a conserved KXXXGXXPWE motif, suggesting that they might recognize the same receptor as LsbB. This notion was supported by the fact that truncated peptides with sequences derived from the C-terminal regions of two LsbB-related bacteriocins inhibited the activity of LsbB, in the same manner as found with the truncated version of LsbB. Taken together, these structure-function studies provide strong evidence that the receptor-binding parts of LsbB and sequence-related bacteriocins are located in their C-terminal halves.
PB  - Amer Soc Biochemistry Molecular Biology Inc, Bethesda
T2  - Journal of Biological Chemistry
T1  - Defining the Structure and Receptor Binding Domain of the Leaderless Bacteriocin LsbB
EP  - 23845
IS  - 34
SP  - 23838
VL  - 289
DO  - 10.1074/jbc.M114.579698
ER  - 

@article{
author = "Ovchinnikov, Kirill V. and Kristiansen, Per E. and Uzelac, Gordana and Topisirović, Ljubiša and Kojić, Milan and Nissen-Meyer, Jon and Nes, Ingolf F. and Diep, Dzung B.",
year = "2014",
abstract = "Background: The bacteriocin LsbB targets a membrane-bound zinc-dependent peptidase. Results: The structure of LsbB was resolved by NMR. The C-terminal unstructured domains of LsbB and several other related bacteriocins were responsible for receptor binding. Conclusion: A subgroup of leaderless bacteriocins has been found to share a similar mechanism in receptor recognition. Significance: The study highlights the structure-function relationship of LsbB. LsbB is a class II leaderless lactococcal bacteriocin of 30 amino acids. In the present work, the structure and function relationship of LsbB was assessed. Structure determination by NMR spectroscopy showed that LsbB has an N-terminal -helix, whereas the C-terminal of the molecule remains unstructured. To define the receptor binding domain of LsbB, a competition assay was performed in which a systematic collection of truncated peptides of various lengths covering different parts of LsbB was used to inhibit the antimicrobial activity of LsbB. The results indicate that the outmost eight-amino acid sequence at the C-terminal end is likely to contain the receptor binding domain because only truncated fragments from this region could antagonize the antimicrobial activity of LsbB. Furthermore, alanine substitution revealed that the tryptophan in position 25 (Trp(25)) is crucial for the blocking activity of the truncated peptides, as well as for the antimicrobial activity of the full-length bacteriocin. LsbB shares significant sequence homology with five other leaderless bacteriocins, especially at their C-terminal halves where all contain a conserved KXXXGXXPWE motif, suggesting that they might recognize the same receptor as LsbB. This notion was supported by the fact that truncated peptides with sequences derived from the C-terminal regions of two LsbB-related bacteriocins inhibited the activity of LsbB, in the same manner as found with the truncated version of LsbB. Taken together, these structure-function studies provide strong evidence that the receptor-binding parts of LsbB and sequence-related bacteriocins are located in their C-terminal halves.",
publisher = "Amer Soc Biochemistry Molecular Biology Inc, Bethesda",
journal = "Journal of Biological Chemistry",
title = "Defining the Structure and Receptor Binding Domain of the Leaderless Bacteriocin LsbB",
pages = "23845-23838",
number = "34",
volume = "289",
doi = "10.1074/jbc.M114.579698"
}

Ovchinnikov, K. V., Kristiansen, P. E., Uzelac, G., Topisirović, L., Kojić, M., Nissen-Meyer, J., Nes, I. F.,& Diep, D. B.. (2014). Defining the Structure and Receptor Binding Domain of the Leaderless Bacteriocin LsbB. in Journal of Biological Chemistry
Amer Soc Biochemistry Molecular Biology Inc, Bethesda., 289(34), 23838-23845.
https://doi.org/10.1074/jbc.M114.579698

Ovchinnikov KV, Kristiansen PE, Uzelac G, Topisirović L, Kojić M, Nissen-Meyer J, Nes IF, Diep DB. Defining the Structure and Receptor Binding Domain of the Leaderless Bacteriocin LsbB. in Journal of Biological Chemistry. 2014;289(34):23838-23845.
doi:10.1074/jbc.M114.579698 .

Ovchinnikov, Kirill V., Kristiansen, Per E., Uzelac, Gordana, Topisirović, Ljubiša, Kojić, Milan, Nissen-Meyer, Jon, Nes, Ingolf F., Diep, Dzung B., "Defining the Structure and Receptor Binding Domain of the Leaderless Bacteriocin LsbB" in Journal of Biological Chemistry, 289, no. 34 (2014):23838-23845,
https://doi.org/10.1074/jbc.M114.579698 . .

TY  - JOUR
AU  - Uzelac, Gordana
AU  - Kojić, Milan
AU  - Lozo, Jelena
AU  - Aleksandrzak-Piekarczyk, Tamara
AU  - Gabrielsen, Christina
AU  - Kristensen, Tom
AU  - Nes, Ingolf F.
AU  - Diep, Dzung B.
AU  - Topisirović, Ljubiša
PY  - 2013
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/703
AB  - Lactococcus lactis subsp. lactis BGMN1-5 produces a leaderless class II bacteriocin called LsbB. To identify the receptor for LsbB, a cosmid library of the LsbB-sensitive strain BGMN1-596 was constructed. About 150 cosmid clones were individually isolated and transferred to LsbB-resistant mutants of BGMN1-596. Cosmid pAZILcos/MN2, carrying a 40-kb insert, was found to restore LsbB sensitivity in LsbB-resistant mutants. Further subcloning revealed that a 1.9-kb fragment, containing only one open reading frame, was sufficient to restore sensitivity. The fragment contains the gene yvjB coding for a Zn-dependent membrane-bound metallopeptidase, suggesting that this gene may serve as the receptor for LsbB. Further support for this notion derives from several independent experiments: (i) whole-genome sequencing confirmed that all LsbB-resistant mutants contain mutations in yvjB; (ii) disruption of yvjB by direct gene knockout rendered sensitive strains BGMN1-596 and IL1403 resistant to LsbB; and (iii) most compellingly, heterologous expression of yvjB in naturally resistant strains of other species, such as Lactobacillus paracasei and Enterococcus faecalis, also rendered them sensitive to the bacteriocin. To our knowledge, this is the first time a membrane-bound peptidase gene has been shown to be involved in bacteriocin sensitivity in target cells. We also demonstrated a novel successful approach for identifying bacteriocin receptors.
PB  - Amer Soc Microbiology, Washington
T2  - Journal of Bacteriology
T1  - A Zn-Dependent Metallopeptidase Is Responsible for Sensitivity to LsbB, a Class II Leaderless Bacteriocin of Lactococcus lactis subsp lactis BGMN1-5
EP  - 5621
IS  - 24
SP  - 5614
VL  - 195
DO  - 10.1128/JB.00859-13
ER  - 

@article{
author = "Uzelac, Gordana and Kojić, Milan and Lozo, Jelena and Aleksandrzak-Piekarczyk, Tamara and Gabrielsen, Christina and Kristensen, Tom and Nes, Ingolf F. and Diep, Dzung B. and Topisirović, Ljubiša",
year = "2013",
abstract = "Lactococcus lactis subsp. lactis BGMN1-5 produces a leaderless class II bacteriocin called LsbB. To identify the receptor for LsbB, a cosmid library of the LsbB-sensitive strain BGMN1-596 was constructed. About 150 cosmid clones were individually isolated and transferred to LsbB-resistant mutants of BGMN1-596. Cosmid pAZILcos/MN2, carrying a 40-kb insert, was found to restore LsbB sensitivity in LsbB-resistant mutants. Further subcloning revealed that a 1.9-kb fragment, containing only one open reading frame, was sufficient to restore sensitivity. The fragment contains the gene yvjB coding for a Zn-dependent membrane-bound metallopeptidase, suggesting that this gene may serve as the receptor for LsbB. Further support for this notion derives from several independent experiments: (i) whole-genome sequencing confirmed that all LsbB-resistant mutants contain mutations in yvjB; (ii) disruption of yvjB by direct gene knockout rendered sensitive strains BGMN1-596 and IL1403 resistant to LsbB; and (iii) most compellingly, heterologous expression of yvjB in naturally resistant strains of other species, such as Lactobacillus paracasei and Enterococcus faecalis, also rendered them sensitive to the bacteriocin. To our knowledge, this is the first time a membrane-bound peptidase gene has been shown to be involved in bacteriocin sensitivity in target cells. We also demonstrated a novel successful approach for identifying bacteriocin receptors.",
publisher = "Amer Soc Microbiology, Washington",
journal = "Journal of Bacteriology",
title = "A Zn-Dependent Metallopeptidase Is Responsible for Sensitivity to LsbB, a Class II Leaderless Bacteriocin of Lactococcus lactis subsp lactis BGMN1-5",
pages = "5621-5614",
number = "24",
volume = "195",
doi = "10.1128/JB.00859-13"
}

Uzelac, G., Kojić, M., Lozo, J., Aleksandrzak-Piekarczyk, T., Gabrielsen, C., Kristensen, T., Nes, I. F., Diep, D. B.,& Topisirović, L.. (2013). A Zn-Dependent Metallopeptidase Is Responsible for Sensitivity to LsbB, a Class II Leaderless Bacteriocin of Lactococcus lactis subsp lactis BGMN1-5. in Journal of Bacteriology
Amer Soc Microbiology, Washington., 195(24), 5614-5621.
https://doi.org/10.1128/JB.00859-13

Uzelac G, Kojić M, Lozo J, Aleksandrzak-Piekarczyk T, Gabrielsen C, Kristensen T, Nes IF, Diep DB, Topisirović L. A Zn-Dependent Metallopeptidase Is Responsible for Sensitivity to LsbB, a Class II Leaderless Bacteriocin of Lactococcus lactis subsp lactis BGMN1-5. in Journal of Bacteriology. 2013;195(24):5614-5621.
doi:10.1128/JB.00859-13 .

Uzelac, Gordana, Kojić, Milan, Lozo, Jelena, Aleksandrzak-Piekarczyk, Tamara, Gabrielsen, Christina, Kristensen, Tom, Nes, Ingolf F., Diep, Dzung B., Topisirović, Ljubiša, "A Zn-Dependent Metallopeptidase Is Responsible for Sensitivity to LsbB, a Class II Leaderless Bacteriocin of Lactococcus lactis subsp lactis BGMN1-5" in Journal of Bacteriology, 195, no. 24 (2013):5614-5621,
https://doi.org/10.1128/JB.00859-13 . .