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Jan Kok - One of the best experts on this subject based on the ideXlab platform.
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Proteins of the lactococcin A secretion system: lcnD encodes two in-frame proteins.
FEMS microbiology letters, 2001Co-Authors: Mario Varcamonti, Gerard Venema, Giovanna Nicastro, Jan KokAbstract:Polyclonal antibodies were raised against LcnC and LcnD proteins of the Lactococcus lactis bacteriocin lactococcin A secretory system to examine their cellular location and interaction. Two major reacting bands were detected by Western immunoblot with the anti-LcnD antibody: one of 52 kDa (LcnD) and another of 45 kDa, called here LcnD*. LcnD* was still detectable after removing the AUG start codon for LcnD. Chemical cross-linking analyses of membrane fractions of L. lactis cells expressing the LcnC/D secretion machinery were performed. Our results indicate that LcnD is present in the secretion machinery complex as a dimer and is able to interact with LcnD* and LcnC.
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Requirement of autolytic activity for bacteriocin-induced lysis
2000Co-Authors: Carmen M. Martínez-cuesta, Jan Kok, Elisabet Herranz, Carmen PeláezAbstract:The bacteriocin produced by Lactococcus lactis IFPL105 is bactericidal against several Lactococcus and Lactobacillus strains. Addition of the bacteriocin to exponential-growth-phase cells resulted in all cases in bacteriolysis. The bacteriolytic response of the strains was not related to differences in sensitivity to the bacteriocin and was strongly reduced in the presence of autolysin inhibitors (Co 2 � and sodium dodecyl sulfate). When L. lactis MG1363 and its derivative deficient in the production of the major autolysin AcmA (MG1363acmA�1) were incubated with the bacteriocin, the latter did not lyse and no intracellular proteins were released into the medium. Incubation of cell wall fragments of L. lactis MG1363, or of L. lactis MG1363acmA�1 to which extracellular AcmA was added, in the presence or absence of the bacteriocin had no effect on the speed of cell wall degradation. This result indicates that the bacteriocin does not degrade cell walls, nor does it directly activate the autolysin AcmA. The autolysin was also responsible for the observed lysis of L. lactis MG1363 cells during incubation with nisin or the mixture of Lactococcins A, B, and M. The results presented here show that lysis of L. lactis after addition of the bacteriocins is caused by the resulting cell damage, which promotes uncontrolled degradation of the cell walls by AcmA. Bacteriocins are antimicrobial polypeptides synthesized ribosomally by bacteria (34). Most bacteriocins from lactic aci
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Membrane topology of the lactococcal bacteriocin ATP-binding cassette transporter protein LcnC. Involvement of LcnC in lactococcin a maturation.
The Journal of biological chemistry, 1999Co-Authors: Christian M. Franke, Gerard Venema, Jan Tiemersma, Jan KokAbstract:Many non-lantibiotic bacteriocins of lactic acid bacteria are produced as precursors with N-terminal leader peptides different from those present in preproteins exported by the general sec-dependent (type II) secretion pathway. These bacteriocins utilize a dedicated (type I) secretion system for externalization. The secretion apparatus for the Lactococcins A, B, and M/N (LcnA, B, and M/N) from Lactococcus lactis is composed of the two membrane proteins LcnC and LcnD. LcnC belongs to the ATP-binding cassette transporters, whereas LcnD is a protein with similarities to other accessory proteins of type I secretion systems. This paper shows that the N-terminal part of LcnC is involved in the processing of the precursor of LcnA. By making translational fusions of LcnC to the reporter proteins beta-galactosidase (LacZ) and alkaline phosphatase (PhoA*), it was shown that both the N- and C-terminal parts of LcnC are located in the cytoplasm. As the N terminus of LcnC is required for LcnA maturation and is localized in the cytoplasm, we conclude that the processing of the bacteriocin LcnA to its mature form takes place at the cytosolic side of the cytoplasmic membrane.
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Rapid and Efficient Purification Method for Small, Hydrophobic, Cationic Bacteriocins: Purification of Lactococcin B and Pediocin PA-1
Applied and environmental microbiology, 1997Co-Authors: Koen Venema, Alfred J. Haandrikman, Gerard Venema, Michael L. Chikindas, Jos F. M. L. Seegers, Kees J. Leenhouts, Jan KokAbstract:The bacteriocins lactococcin B and pediocin PA-1 were purified by ethanol precipitation, preparative isoelectric focusing, and ultrafiltration. The procedure reproducibly leads to high final yields in comparison to the generally low yields obtained by column chromatography. Specifically, during isoelectric focusing no loss of activity occurs. The method, in general, should be applicable to small, hydrophobic, cationic bacteriocins.
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Mutational analysis and chemical modification of Cys24 of lactococcin B, a bacteriocin produced by Lactococcus lactis
Microbiology, 1996Co-Authors: Koen Venema, Michiel H. R. Dost, Gerard Venema, Jan KokAbstract:Using site-directed mutagenesis the single cysteine residue at position 24 of lactococcin B was replaced by all other possible amino acids. Most of these mutant molecules retained bacteriocin activity, with the exception of those in which cysteine was replaced by a positively charged amino acid. This would seem to be in agreement with the authors' earlier observation that treatment of the wild-type molecule with HgCI2 resulted in its inactivation. The factor that causes inactivation of lactococcin B seems to be the introduction of a positive charge at position 24 by HgCI2 rather than oxidation of this residue, as treatment of the bacteriocin with other oxidative chemicals did not interfere with the ability of lactococcin B to dissipate the membrane potential of sensitive cells. Results are also reported which imply that inactive lactococcin B can still bind to its receptor. It can be replaced by an active bacteriocin molecule, resulting in dissipation of the membrane potential.
Kenji Sonomoto - One of the best experts on this subject based on the ideXlab platform.
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Processing and secretion of non-cognate bacteriocins by EnkT, an ABC transporter from a multiple-bacteriocin producer, Enterococcus faecium NKR-5-3.
Journal of bioscience and bioengineering, 2020Co-Authors: Hirotoshi Sushida, Naoki Ishibashi, Takeshi Zendo, Jiro Nakayama, Miyuki Sakei, Rodney H. Perez, Pongtep Wilaipun, Vichien Leelawatcharamas, Kenji SonomotoAbstract:EnkT is an ATP-binding cassette (ABC) transporter produced by Enterococcus faecium NKR-5-3, which is responsible for the secretion of multiple bacteriocins; enterocins NKR-5-3A, C, D, and Z (Ent53A, C, D, and Z). EnkT has been shown to possess a tolerant recognition mechanism that enables it to secrete the mature Ent53C from a chimeric precursor peptide containing the leader peptide moieties that are derived from different heterologous bacteriocins. In this study, to further characterize EnkT, we aimed to investigate the capacity of EnkT to recognize, process, and secrete non-cognate bacteriocins, which belong to different subclasses of class II. For this, the non-cognate bacteriocin precursor peptides, including enterocin A, pediocin PA-1, lactococcin Q, lactococcin A, and lacticin Q were co-expressed with EnkT, and thereafter, the production of the mature forms of these non-cognate bacteriocins was assessed. Our results revealed that EnkT could potentially recognize, process, and secrete the non-cognate bacteriocins with an exception of the leaderless bacteriocin, lacticin Q. Moreover, the processing and secretion efficiencies of these heterologous non-cognate bacteriocins by EnkT were further enhanced when the leader peptide moiety was replaced with the Ent53C leader peptide (derived from a native NKR-5-3 bacteriocin). The findings of this study describe the wide substrate tolerance of this ABC transporter, EnkT, that can be exploited in the future in establishing effective bacteriocin production systems adaptive to complex fermentation conditions common in many food systems.
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Characterisation of the action mechanism of a Lactococcus-specific bacteriocin, lactococcin Z.
Journal of bioscience and bioengineering, 2018Co-Authors: Ghoson M Daba, Naoki Ishibashi, Takeshi Zendo, Xiao Gong, Hiroya Taki, Keisuke Yamashiro, Yen Yi Lim, Kenji SonomotoAbstract:Lactococcin Z is a novel Lactococcus-specific bacteriocin produced by Lactococcus lactis QU 7 that shares 55.6% identity with lactococcin A. To identify the receptor targeted by lactococcin Z, several lactococcin Z-resistant mutants were generated from the sensitive strain, L. lactis IL1403. The resistant mutants showed difficulties in utilising mannose and glucose as sole carbon sources, contrary to their pattern of growth in the presence of galactose as a sole carbon source. Mutations were found in the ptnC and ptnD genes of lactococcin Z-resistant mutants, which encode the mannose phosphotransferase system (Man-PTS) components, IIC and IID, respectively; therefore, IIC and IID are proposed as potential receptors employed by lactococcin Z and are the same receptors targeted by lactococcin A. Both Lactococcins A and Z share a high percentage identity in their N-termini regions in contrast to their C-termini that show less similarity; this may explain the difference in their action mechanisms as well as the lack of cross-immunity between them. Although lactococcin Z showed bactericidal activity, it neither dissipated membrane potential nor formed pores on the membranes of sensitive cells, in sharp contrast to the pore-forming lactococcin A.
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Functional analysis of the biosynthetic gene cluster required for immunity and secretion of a novel Lactococcus-specific bacteriocin, lactococcin Z.
Journal of applied microbiology, 2017Co-Authors: Ghoson M Daba, Naoki Ishibashi, Takeshi Zendo, Kenji SonomotoAbstract:Aims Characterization of the biosynthesis (secretion and immunity) of lactococcin Z. Methods and Results Lactococcin Z is produced by Lactococcus lactisQU 7. DNA sequence analysis revealed that the lactococcin Z gene cluster (c. 5·1 kb) includes four genes encoding putative biosynthetic proteins, LczB (self-immunity protein), LczC (an ABC transporter) and LczD (a transport accessory protein), besides the previously identified LczA. LczB showed 25·5% identity to LciA, the lactococcin A immunity protein, while LczC and LczD had 93·7 and 95·3% identities, respectively, to corresponding proteins of lactococcin A. Heterologous expression of various combinations of the four genes indicated that lczB confers self-immunity against lactococcin Z, and that the four genes are necessary to produce lactococcin Z. However, LczB and LciA showed no cross-immunity to Lactococcins A and Z respectively. Conclusions The results verified that LczB is the lactococcin Z immunity protein, and LczC is responsible for lactococcin Z secretion in a manner dependent on LczD expression. Significance and Impact of the Study The biosynthesis (secretion and immunity) of a new Lactococcus-specific bacteriocin, lactococcin Z, was characterized. Moreover, the results suggested that lactococcin Z has different immunity and action mechanisms from other Lactococcus-specific bacteriocins.
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Identification of Lactococcus-Specific Bacteriocins Produced by Lactococcal Isolates, and the Discovery of a Novel Bacteriocin, Lactococcin Z
Probiotics and Antimicrobial Proteins, 2015Co-Authors: Naoki Ishibashi, Takeshi Zendo, Shoko Koga, Hiromi Seto, Kenji SonomotoAbstract:Lactic acid bacteria that produce Lactococcus -specific bacteriocins were isolated and identified as Lactococcus lactis from fresh corn or lettuce. Among them, four isolates were identified as lactococcin Q producers. Seven isolates showed antimicrobial activity against a lactococcin Q producer, L. lactis QU 4, as well as against nisin Z and lacticin Q producers belonging to L. lactis . Strain QU 7 was selected as a standard strain and showed no cross-immunity to lactococcin Q or other lactococcal bacteriocins. The bacteriocin produced by strain QU 7 was purified in three chromatographic steps, and its molecular mass was determined to be 5041.35 Da. The amino acid sequence analysis revealed that it is a novel class IId bacteriocin, referred to as lactococcin Z. It consisted of 45 amino acid residues. The lczA gene encoding the prepeptide of lactococcin Z showed homology to Lactococcins A, B, and M. Thus, this report demonstrates a new example of Lactococcus -specific bacteriocins.
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Molecular characterization of the genes involved in the secretion and immunity of lactococcin Q, a two-peptide bacteriocin produced by Lactococcus lactis QU 4.
Microbiology (Reading England), 2015Co-Authors: Naoki Ishibashi, Takeshi Zendo, Shoko Koga, Yasushi Shigeri, Kenji SonomotoAbstract:Lactococcin Q is a two-peptide (Qα and Qβ) bacteriocin produced by Lactococcus lactis QU 4, which exhibits specific antimicrobial activity against L. lactis strains. The lactococcin Q gene cluster (approximately 4.5 kb) was sequenced and found to include genes encoding lactococcin Q immunity (laqC), an ATP-binding cassette transporter (laqD) and a transport accessory protein (laqE), downstream of the lactococcin Q structural genes (laqA and laqB). In addition, the gene cluster showed high sequence identity with that of a lactococcin Q homologue bacteriocin, lactococcin G. Heterologous expression studies showed that LaqD was responsible for lactococcin Q secretion in a manner dependent on LaqE expression, and that LaqC conferred self-immunity to lactococcin Q and cross-immunity to lactococcin G. Amino acid alignment of both lactococcin transporters revealed that LaqD contains an insertion (160-168 residues) that is essential for lactococcin Q secretion, as L. lactis cells that expressed LaqDΔ160-168 were devoid of this function. Additional experiments demonstrated that the LaqDΔ160-168 mutant was, however, able to secrete lactococcin G, suggesting that the insertion is necessary only for the lactococcin Q secretion by LaqD. This report demonstrates the biosynthetic mechanism of lactococcin Q/G-type bacteriocins and the complementarity of the genes responsible for the secretion of Lactococcins Q and G.
Dzung B. Diep - One of the best experts on this subject based on the ideXlab platform.
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The extracellular loop of Man-PTS subunit IID is responsible for the sensitivity of Lactococcus garvieae to garvicins A, B and C.
Scientific reports, 2018Co-Authors: Aleksandra Tymoszewska, Dzung B. Diep, Tamara Aleksandrzak-piekarczykAbstract:Mannose phosphotransferase system (Man-PTS) serves as a receptor for several bacteriocins in sensitive bacterial cells, namely subclass IIa bacteriocins (pediocin-like; pediocins) and subclass IId ones - lactococcin A (LcnA), lactococcin B (LcnB) and garvicin Q (GarQ). Here, to identify the receptor for three other narrow-spectrum subclass IId bacteriocins - garvicins A, B and C (GarA-C) Lactococcus garvieae mutants resistant to bacteriocins were generated and sequenced to look for mutations responsible for resistance. Spontaneous mutants had their whole genome sequenced while in mutants obtained by integration of pGhost9::ISS1 regions flanking the integration site were sequenced. For both types of mutants mutations were found in genes encoding Man-PTS components IIC and IID indicating that Man-PTS likely serves as the receptor for these bacteriocins as well. This was subsequently confirmed by deletion of the man-PTS operon in the bacteriocin-sensitive L. garvieae IBB3403, which resulted in resistant cells, and by heterologous expression of appropriate man-PTS genes in the resistant Lactococcus lactis strains, which resulted in sensitive cells. GarA, GarB, GarC and other Man-PTS-targeting bacteriocins differ in the amino acid sequence and activity spectrum, suggesting that they interact with the receptor through distinct binding patterns. Comparative analyses and genetic studies identified a previously unrecognized extracellular loop of Man-PTS subunit IID (γ+) implicated in the L. garvieae sensitivity to the bacteriocins studied here. Additionally, individual amino acids localized mostly in the sugar channel-forming transmembrane parts of subunit IIC or in the extracellular parts of IID likely involved in the interaction with each bacteriocin were specified. Finally, template-based 3D models of Man-PTS subunits IIC and IID were built to allow a deeper insight into the Man-PTS structure and functioning.
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The Non-Lantibiotic Bacteriocin Garvicin Q Targets Man-PTS in a Broad Spectrum of Sensitive Bacterial Genera
Scientific reports, 2017Co-Authors: Aleksandra Tymoszewska, Dzung B. Diep, Paulina Wirtek, Tamara Aleksandrzak-piekarczykAbstract:Mannose phosphotransferase system (Man-PTS) is the main mannose permease in bacteria but it is also a known receptor for subclass IIa bacteriocins (pediocin-like group) as well as subclass IId lactococcin A (LcnA) and lactococcin B (LcnB) (LcnA-like group). Subclass IIa bacteriocins exhibit a strong activity against Listeria spp. but they are not against Lactococcus spp. In contrast, the LcnA-like bacteriocins act only against Lactococcus lactis strains. Garvicin Q (GarQ) is a subclass IId bacteriocin with minor similarity to LcnA-like bacteriocins and a relatively broad antimicrobial spectrum including, among others, Listeria and Lactococcus spp. To identify the GarQ receptor, we obtained GarQ-resistant mutants of Lactococcus garvieae IBB3403 and L. lactis IL1403 and sequenced their genomes that revealed mutations in genes encoding the membrane-bound Man-PTS IIC or IID subunits encoded by ptnCD in L. lactis and manCD in L. garvieae. This is the first time that a bacteriocin outside the pediocin- and LcnA-like groups is shown to target Man-PTS. The interaction between GarQ and Man-PTS may occur through a new binding pattern involving specific amino acids highly conserved among the GarQ-sensitive bacterial species located in the N-terminal part and extracellular loops of subunit IID and in transmembrane region of IIC.
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Sensitivity to the two‐peptide bacteriocin lactococcin G is dependent on UppP, an enzyme involved in cell‐wall synthesis
Molecular microbiology, 2014Co-Authors: Morten Kjos, Camilla Oppegård, Dzung B. Diep, Jon Nissen-meyer, Ingolf F Nes, Jan-willem Veening, Tom KristensenAbstract:Most bacterially produced antimicrobial peptides (bacteriocins) are thought to kill target cells by a receptor-mediated mechanism. However, for most bacteriocins the receptor is unknown. For instance, no target receptor has been identified for the two-peptide bacteriocins (class IIb), whose activity requires the combined action of two individual peptides. To identify the receptor for the class IIb bacteriocin lactococcin G, which targets strains of Lactococcus lactis, we generated 12 lactococcin G-resistant mutants and performed whole-genome sequencing to identify mutations causing the resistant phenotype. Remarkably, all had a mutation in or near the gene uppP (bacA), encoding an undecaprenyl pyrophosphate phosphatase; a membrane protein involved in peptidoglycan synthesis. Nine mutants had stop codons or frameshifts in the uppP gene, two had point mutations in putative regulatory regions and one caused an amino acid substitution in UppP. To verify the receptor function of UppP, it was shown that growth of non-sensitive Streptococcus pneumoniae could be inhibited by lactococcin G when L. lactis uppP was expressed in this bacterium. Furthermore, we show that the related class IIb bacteriocin enterocin 1071 also uses UppP as receptor. The approach used here should be broadly applicable to identify receptors for other bacteriocins as well.
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mechanisms of resistance to bacteriocins targeting the mannose phosphotransferase system
Applied and Environmental Microbiology, 2011Co-Authors: Morten Kjos, Ingolf F Nes, Dzung B. DiepAbstract:The membrane proteins IIC and IID of the mannose phosphotransferase system (Man-PTS) together form a membrane-located complex that serves as a receptor for several different bacteriocins, including the pediocin-like class IIa bacteriocins and the class IIc bacteriocin lactococcin A. Bacterial strains sensitive to class IIa bacteriocins readily give rise to resistant mutants upon bacteriocin exposure. In the present study, we have therefore investigated lactococcin A-resistant mutants of Lactococcus lactis as well as natural food isolates of Listeria monocytogenes with different susceptibilities to class IIa bacteriocins. We found two major mechanisms of resistance. The first involves downregulation of Man-PTS gene expression, which takes place both in spontaneous resistant mutants and in natural resistant isolates. The second involves normal expression of the Man-PTS system, but the underlying mechanism of resistance for these cells is unknown. In some cases, the resistant phenotype was linked to a shift in the metabolism; i.e., reduced growth on glucose due to reduction in Man-PTS expression was accompanied by enhanced growth on another sugar, such as galactose. The implications of these findings in terms of metabolic heterogeneity are discussed.
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an extracellular loop of the mannose phosphotransferase system component iic is responsible for specific targeting by class iia bacteriocins
Journal of Bacteriology, 2010Co-Authors: Morten Kjos, Zhian Salehian, Ingolf F Nes, Dzung B. DiepAbstract:Bacteriocins are small, ribosomally synthesized antimicrobial peptides that normally kill bacteria closely related to the bacteriocin producers, but some also target a wider spectrum of bacteria, including a number of pathogens and food spoilage bacterial species (5, 28). Class IIa (pediocin-like) bacteriocins display a broad antimicrobial spectrum, including important pathogens such as Listeria monocytogenes and Enterococcus faecalis. These peptides consist of 37 to 48 nonmodified amino acids, contain a conserved pediocin-box sequence (Y-G-N-G-V/L) in the N-terminal region, and have defined secondary features in their structure: a cationic β sheet at the conserved N terminus and a helix-containing domain at the less-conserved C terminus (16, 30). Class IIa bacteriocins target sensitive cells by using the mannose phosphotransferase system (man-PTS) as a receptor (6, 10, 17, 19, 33). This sugar uptake system is the major glucose transporter for many bacteria, particularly Firmicutes and Gammaproteobacteria (39). Each man-PTS complex consists of four structural domains: IIC and IID, represented by two membrane-located proteins, and IIA and IIB, which are normally represented by a single cytoplasmic protein that can form reversible contacts with its membrane-located partners (31). It has previously been shown that coexpression of the IIC and IID genes is needed to confer sensitivity to class IIa bacteriocins as well as to the lactococcal bacteriocin lactococcin A and that the cytoplasmic IIAB partner is not involved in this process (10). However, while lactococcin A (belonging to class IIc) targets only the lactococcal man-PTS, the class IIa bacteriocins target man-PTSs of species of diverse genera (e.g., Listeria, Enterococcus, and Lactobacillus) but somehow not those of the Lactococcus genus (24). This genus specificity has been recognized for almost 2 decades (20, 23, 26); still, the molecular nature underlying the specificity has remained very enigmatic. In the present report we clarify this issue by demonstrating that these two types of bacteriocins exhibit different binding patterns on their receptors: class IIa bacteriocins specifically interact with a defined region of 40 amino acids in the IIC protein whereas lactococcin A has a more complex interaction involving regions from both IIC and IID.
Jon Nissen-meyer - One of the best experts on this subject based on the ideXlab platform.
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Nuclear Magnetic Resonance Structure and Mutational Analysis of the Lactococcin A Immunity Protein
Biochemistry, 2016Co-Authors: Per Eugen Kristiansen, Virginia Fuochi, Anders Pedersen, Jon Nissen-meyer, Goran Karlsson, Cecilia Persson, Camilla OppegårdAbstract:The class IId bacteriocin lactococcin A and the pediocin-like bacteriocins induce membrane leakage and cell death by specifically binding the mannose phophotransferase system (man-PTS) on their target cells. The bacteriocins’ cognate immunity proteins that protect the producer cell from its own bacteriocin recognize and bind to the bacteriocin–man-PTS complex and thereby block membrane leakage. In this study, we have determined the three-dimensional structure of the lactococcin A immunity protein (LciA) by the use of nuclear magnetic resonance spectroscopy. LciA forms a four-helix bundle structure with a flexible C-terminal tail. Despite the low degree of sequence similarity between LciA and the pediocin-like immunity proteins, they share the same fold. However, there are certain differences between the structures. The C-terminal helix in LciA is considerably shorter than that observed in the pediocin-like immunity proteins, and the surface potentials of the immunity proteins differ. Truncated variants of...
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Sensitivity to the two‐peptide bacteriocin lactococcin G is dependent on UppP, an enzyme involved in cell‐wall synthesis
Molecular microbiology, 2014Co-Authors: Morten Kjos, Camilla Oppegård, Dzung B. Diep, Jon Nissen-meyer, Ingolf F Nes, Jan-willem Veening, Tom KristensenAbstract:Most bacterially produced antimicrobial peptides (bacteriocins) are thought to kill target cells by a receptor-mediated mechanism. However, for most bacteriocins the receptor is unknown. For instance, no target receptor has been identified for the two-peptide bacteriocins (class IIb), whose activity requires the combined action of two individual peptides. To identify the receptor for the class IIb bacteriocin lactococcin G, which targets strains of Lactococcus lactis, we generated 12 lactococcin G-resistant mutants and performed whole-genome sequencing to identify mutations causing the resistant phenotype. Remarkably, all had a mutation in or near the gene uppP (bacA), encoding an undecaprenyl pyrophosphate phosphatase; a membrane protein involved in peptidoglycan synthesis. Nine mutants had stop codons or frameshifts in the uppP gene, two had point mutations in putative regulatory regions and one caused an amino acid substitution in UppP. To verify the receptor function of UppP, it was shown that growth of non-sensitive Streptococcus pneumoniae could be inhibited by lactococcin G when L. lactis uppP was expressed in this bacterium. Furthermore, we show that the related class IIb bacteriocin enterocin 1071 also uses UppP as receptor. The approach used here should be broadly applicable to identify receptors for other bacteriocins as well.
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Structure analysis of the two-peptide bacteriocin lactococcin G by introducing D-amino acid residues
Microbiology, 2010Co-Authors: Camilla Oppegård, Per Eugen Kristiansen, Per Rogne, Jon Nissen-meyerAbstract:The importance of 3D structuring in the N- and C-terminal ends of the two peptides (39-mer LcnG-α and 35-mer LcnG-β) that constitute the two-peptide bacteriocin lactococcin G was analysed by replacing residues in the end regions with the corresponding d-isomeric residues. When assayed for antibacterial activity in combination with the complementary wild-type peptide, LcnG-α with four d-residues in its C-terminal region and LcnG-β with four d-residues in either its N- or its C-terminal region were relatively active (two- to 20-fold reduction in activity). 3D structuring of the C-terminal region in LcnG-α and the C- and N-terminal regions in LcnG-β is thus not particularly critical for retaining antibacterial activity, indicating that the 3D structure of these regions is not vital for interpeptide interactions or for interactions between the peptides and cellular components. The 3D structure of the N-terminal region in LcnG-α may be more important, as LcnG-α with four N-terminal d-residues was the least active of these four peptides (10- to 100-fold reduction in activity). The results are consistent with a proposed structural model of lactococcin G in which LcnG-α and -β form a transmembrane parallel helix–helix structure involving approximately 20 residues in each peptide, starting near the N terminus of LcnG-α and at about residue 13 in LcnG-β. Upon expressing the lactococcin G immunity protein, sensitive target cells became resistant to all of these d-residue-containing peptides. The end regions of the two lactococcin G peptides are consequently not involved in essential structure-dependent interactions with the immunity protein. The relatively high activity of most of the d-residue-containing peptides suggests that bacteriocins with increased resistance to exopeptidases may be generated by replacing their N- and C-terminal residues with d-residues.
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Structure analysis of the two-peptide bacteriocin lactococcin G by introducing D-amino acid residues.
Microbiology (Reading England), 2010Co-Authors: Camilla Oppegård, Per Eugen Kristiansen, Per Rogne, Jon Nissen-meyerAbstract:The importance of 3D structuring in the N- and C-terminal ends of the two peptides (39-mer LcnG-alpha and 35-mer LcnG-beta) that constitute the two-peptide bacteriocin lactococcin G was analysed by replacing residues in the end regions with the corresponding D-isomeric residues. When assayed for antibacterial activity in combination with the complementary wild-type peptide, LcnG-alpha with four D-residues in its C-terminal region and LcnG-beta with four d-residues in either its N- or its C-terminal region were relatively active (two- to 20-fold reduction in activity). 3D structuring of the C-terminal region in LcnG-alpha and the C- and N-terminal regions in LcnG-beta is thus not particularly critical for retaining antibacterial activity, indicating that the 3D structure of these regions is not vital for interpeptide interactions or for interactions between the peptides and cellular components. The 3D structure of the N-terminal region in LcnG-alpha may be more important, as LcnG-alpha with four N-terminal D-residues was the least active of these four peptides (10- to 100-fold reduction in activity). The results are consistent with a proposed structural model of lactococcin G in which LcnG-alpha and -beta form a transmembrane parallel helix-helix structure involving approximately 20 residues in each peptide, starting near the N terminus of LcnG-alpha and at about residue 13 in LcnG-beta. Upon expressing the lactococcin G immunity protein, sensitive target cells became resistant to all of these D-residue-containing peptides. The end regions of the two lactococcin G peptides are consequently not involved in essential structure-dependent interactions with the immunity protein. The relatively high activity of most of the D-residue-containing peptides suggests that bacteriocins with increased resistance to exopeptidases may be generated by replacing their N- and C-terminal residues with d-residues.
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The Lactococcin G Immunity Protein Recognizes Specific Regions in Both Peptides Constituting the Two-Peptide Bacteriocin Lactococcin G
Applied and environmental microbiology, 2009Co-Authors: Camilla Oppegård, Linda Emanuelsen, Lisbeth Thorbek, Gunnar Fimland, Jon Nissen-meyerAbstract:Lactococcin G and enterocin 1071 are two homologous two-peptide bacteriocins. Expression vectors containing the gene encoding the putative lactococcin G immunity protein (lagC) or the gene encoding the enterocin 1071 immunity protein (entI) were constructed and introduced into strains sensitive to one or both of the bacteriocins. Strains that were sensitive to lactococcin G became immune to lactococcin G when expressing the putative lactococcin G immunity protein, indicating that the lagC gene in fact encodes a protein involved in lactococcin G immunity. To determine which peptide or parts of the peptide(s) of each bacteriocin that are recognized by the cognate immunity protein, combinations of wild-type peptides and hybrid peptides from the two bacteriocins were assayed against strains expressing either of the two immunity proteins. The lactococcin G immunity protein rendered the enterococcus strain but not the lactococcus strains resistant to enterocin 1071, indicating that the functionality of the immunity protein depends on a cellular component. Moreover, regions important for recognition by the immunity protein were identified in both peptides (Lcn-α and Lcn-β) constituting lactococcin G. These regions include the N-terminal end of Lcn-α (residues 1 to 13) and the C-terminal part of Lcn-β (residues 14 to 24). According to a previously proposed structural model of lactococcin G, these regions will be positioned adjacent to each other in the transmembrane helix-helix structure, and the model thus accommodates the present results.
Takeshi Zendo - One of the best experts on this subject based on the ideXlab platform.
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Processing and secretion of non-cognate bacteriocins by EnkT, an ABC transporter from a multiple-bacteriocin producer, Enterococcus faecium NKR-5-3.
Journal of bioscience and bioengineering, 2020Co-Authors: Hirotoshi Sushida, Naoki Ishibashi, Takeshi Zendo, Jiro Nakayama, Miyuki Sakei, Rodney H. Perez, Pongtep Wilaipun, Vichien Leelawatcharamas, Kenji SonomotoAbstract:EnkT is an ATP-binding cassette (ABC) transporter produced by Enterococcus faecium NKR-5-3, which is responsible for the secretion of multiple bacteriocins; enterocins NKR-5-3A, C, D, and Z (Ent53A, C, D, and Z). EnkT has been shown to possess a tolerant recognition mechanism that enables it to secrete the mature Ent53C from a chimeric precursor peptide containing the leader peptide moieties that are derived from different heterologous bacteriocins. In this study, to further characterize EnkT, we aimed to investigate the capacity of EnkT to recognize, process, and secrete non-cognate bacteriocins, which belong to different subclasses of class II. For this, the non-cognate bacteriocin precursor peptides, including enterocin A, pediocin PA-1, lactococcin Q, lactococcin A, and lacticin Q were co-expressed with EnkT, and thereafter, the production of the mature forms of these non-cognate bacteriocins was assessed. Our results revealed that EnkT could potentially recognize, process, and secrete the non-cognate bacteriocins with an exception of the leaderless bacteriocin, lacticin Q. Moreover, the processing and secretion efficiencies of these heterologous non-cognate bacteriocins by EnkT were further enhanced when the leader peptide moiety was replaced with the Ent53C leader peptide (derived from a native NKR-5-3 bacteriocin). The findings of this study describe the wide substrate tolerance of this ABC transporter, EnkT, that can be exploited in the future in establishing effective bacteriocin production systems adaptive to complex fermentation conditions common in many food systems.
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Characterisation of the action mechanism of a Lactococcus-specific bacteriocin, lactococcin Z.
Journal of bioscience and bioengineering, 2018Co-Authors: Ghoson M Daba, Naoki Ishibashi, Takeshi Zendo, Xiao Gong, Hiroya Taki, Keisuke Yamashiro, Yen Yi Lim, Kenji SonomotoAbstract:Lactococcin Z is a novel Lactococcus-specific bacteriocin produced by Lactococcus lactis QU 7 that shares 55.6% identity with lactococcin A. To identify the receptor targeted by lactococcin Z, several lactococcin Z-resistant mutants were generated from the sensitive strain, L. lactis IL1403. The resistant mutants showed difficulties in utilising mannose and glucose as sole carbon sources, contrary to their pattern of growth in the presence of galactose as a sole carbon source. Mutations were found in the ptnC and ptnD genes of lactococcin Z-resistant mutants, which encode the mannose phosphotransferase system (Man-PTS) components, IIC and IID, respectively; therefore, IIC and IID are proposed as potential receptors employed by lactococcin Z and are the same receptors targeted by lactococcin A. Both Lactococcins A and Z share a high percentage identity in their N-termini regions in contrast to their C-termini that show less similarity; this may explain the difference in their action mechanisms as well as the lack of cross-immunity between them. Although lactococcin Z showed bactericidal activity, it neither dissipated membrane potential nor formed pores on the membranes of sensitive cells, in sharp contrast to the pore-forming lactococcin A.
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Functional analysis of the biosynthetic gene cluster required for immunity and secretion of a novel Lactococcus-specific bacteriocin, lactococcin Z.
Journal of applied microbiology, 2017Co-Authors: Ghoson M Daba, Naoki Ishibashi, Takeshi Zendo, Kenji SonomotoAbstract:Aims Characterization of the biosynthesis (secretion and immunity) of lactococcin Z. Methods and Results Lactococcin Z is produced by Lactococcus lactisQU 7. DNA sequence analysis revealed that the lactococcin Z gene cluster (c. 5·1 kb) includes four genes encoding putative biosynthetic proteins, LczB (self-immunity protein), LczC (an ABC transporter) and LczD (a transport accessory protein), besides the previously identified LczA. LczB showed 25·5% identity to LciA, the lactococcin A immunity protein, while LczC and LczD had 93·7 and 95·3% identities, respectively, to corresponding proteins of lactococcin A. Heterologous expression of various combinations of the four genes indicated that lczB confers self-immunity against lactococcin Z, and that the four genes are necessary to produce lactococcin Z. However, LczB and LciA showed no cross-immunity to Lactococcins A and Z respectively. Conclusions The results verified that LczB is the lactococcin Z immunity protein, and LczC is responsible for lactococcin Z secretion in a manner dependent on LczD expression. Significance and Impact of the Study The biosynthesis (secretion and immunity) of a new Lactococcus-specific bacteriocin, lactococcin Z, was characterized. Moreover, the results suggested that lactococcin Z has different immunity and action mechanisms from other Lactococcus-specific bacteriocins.
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Identification of Lactococcus-Specific Bacteriocins Produced by Lactococcal Isolates, and the Discovery of a Novel Bacteriocin, Lactococcin Z
Probiotics and Antimicrobial Proteins, 2015Co-Authors: Naoki Ishibashi, Takeshi Zendo, Shoko Koga, Hiromi Seto, Kenji SonomotoAbstract:Lactic acid bacteria that produce Lactococcus -specific bacteriocins were isolated and identified as Lactococcus lactis from fresh corn or lettuce. Among them, four isolates were identified as lactococcin Q producers. Seven isolates showed antimicrobial activity against a lactococcin Q producer, L. lactis QU 4, as well as against nisin Z and lacticin Q producers belonging to L. lactis . Strain QU 7 was selected as a standard strain and showed no cross-immunity to lactococcin Q or other lactococcal bacteriocins. The bacteriocin produced by strain QU 7 was purified in three chromatographic steps, and its molecular mass was determined to be 5041.35 Da. The amino acid sequence analysis revealed that it is a novel class IId bacteriocin, referred to as lactococcin Z. It consisted of 45 amino acid residues. The lczA gene encoding the prepeptide of lactococcin Z showed homology to Lactococcins A, B, and M. Thus, this report demonstrates a new example of Lactococcus -specific bacteriocins.
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Molecular characterization of the genes involved in the secretion and immunity of lactococcin Q, a two-peptide bacteriocin produced by Lactococcus lactis QU 4.
Microbiology (Reading England), 2015Co-Authors: Naoki Ishibashi, Takeshi Zendo, Shoko Koga, Yasushi Shigeri, Kenji SonomotoAbstract:Lactococcin Q is a two-peptide (Qα and Qβ) bacteriocin produced by Lactococcus lactis QU 4, which exhibits specific antimicrobial activity against L. lactis strains. The lactococcin Q gene cluster (approximately 4.5 kb) was sequenced and found to include genes encoding lactococcin Q immunity (laqC), an ATP-binding cassette transporter (laqD) and a transport accessory protein (laqE), downstream of the lactococcin Q structural genes (laqA and laqB). In addition, the gene cluster showed high sequence identity with that of a lactococcin Q homologue bacteriocin, lactococcin G. Heterologous expression studies showed that LaqD was responsible for lactococcin Q secretion in a manner dependent on LaqE expression, and that LaqC conferred self-immunity to lactococcin Q and cross-immunity to lactococcin G. Amino acid alignment of both lactococcin transporters revealed that LaqD contains an insertion (160-168 residues) that is essential for lactococcin Q secretion, as L. lactis cells that expressed LaqDΔ160-168 were devoid of this function. Additional experiments demonstrated that the LaqDΔ160-168 mutant was, however, able to secrete lactococcin G, suggesting that the insertion is necessary only for the lactococcin Q secretion by LaqD. This report demonstrates the biosynthetic mechanism of lactococcin Q/G-type bacteriocins and the complementarity of the genes responsible for the secretion of Lactococcins Q and G.
