Abstract:Post-translationally modified, (methyl-)lanthionine-containing peptides are produced by several Gram-positive bacteria. These so-called lantibiotics have potent activity against various bacterial pathogens including multidrug-resistant strains and are thus discussed as alternatives to antibiotics. Several naturally occurring mechanisms of resistance against lantibiotics have been described for bacteria, including cell envelope modifications, ABC-transporters, lipoproteins and peptidases. Corynebacterium specie… Show more
“…Although previous studies reported C. glutamicum to be resistant to up to 40 µg/ml of nisin [ 46 ], our own results suggest that growth of C. glutamicum is completely inhibited by ~ 1 µg/ml of nisin [ 47 , 48 ]. To corroborate these results and to determine the nisin concentration that is required for formation of pores in the membrane of C. glutamicum , we devised a biosensor expressing the pH-dependent fluorescent protein pHluorin2 [ 49 ] in a similar fashion as described recently for L. monocytogenes [ 50 ].…”
Section: Resultscontrasting
confidence: 70%
“…1 A). This indicates that recombinant production of active nisin is not possible using C. glutamicum as the product not only inhibits growth [ 47 , 48 ] but kills producer cells at low concentrations. Fig.…”
Background
The bacteriocin nisin is naturally produced by Lactococcus lactis as an inactive prepeptide that is modified posttranslationally resulting in five (methyl-)lanthionine rings characteristic for class Ia bacteriocins. Export and proteolytic cleavage of the leader peptide results in release of active nisin. By targeting the universal peptidoglycan precursor lipid II, nisin has a broad target spectrum including important human pathogens such as Listeria monocytogenes and methicillin-resistant Staphylococcus aureus strains. Industrial nisin production is currently performed using natural producer strains resulting in rather low product purity and limiting its application to preservation of dairy food products.
Results
We established heterologous nisin production using the biotechnological workhorse organism Corynebacterium glutamicum in a two-step process. We demonstrate successful biosynthesis and export of fully modified prenisin and its activation to mature nisin by a purified, soluble variant of the nisin protease NisP (sNisP) produced in Escherichia coli. Active nisin was detected by a L. lactis sensor strain with strictly nisin-dependent expression of the fluorescent protein mCherry. Following activation by sNisP, supernatants of the recombinant C. glutamicum producer strain cultivated in standard batch fermentations contained at least 1.25 mg/l active nisin.
Conclusions
We demonstrate successful implementation of a two-step process for recombinant production of active nisin with C. glutamicum. This extends the spectrum of bioactive compounds that may be produced using C. glutamicum to a bacteriocin harboring complex posttranslational modifications. Our results provide a basis for further studies to optimize product yields, transfer production to sustainable substrates and purification of pharmaceutical grade nisin.
“…Although previous studies reported C. glutamicum to be resistant to up to 40 µg/ml of nisin [ 46 ], our own results suggest that growth of C. glutamicum is completely inhibited by ~ 1 µg/ml of nisin [ 47 , 48 ]. To corroborate these results and to determine the nisin concentration that is required for formation of pores in the membrane of C. glutamicum , we devised a biosensor expressing the pH-dependent fluorescent protein pHluorin2 [ 49 ] in a similar fashion as described recently for L. monocytogenes [ 50 ].…”
Section: Resultscontrasting
confidence: 70%
“…1 A). This indicates that recombinant production of active nisin is not possible using C. glutamicum as the product not only inhibits growth [ 47 , 48 ] but kills producer cells at low concentrations. Fig.…”
Background
The bacteriocin nisin is naturally produced by Lactococcus lactis as an inactive prepeptide that is modified posttranslationally resulting in five (methyl-)lanthionine rings characteristic for class Ia bacteriocins. Export and proteolytic cleavage of the leader peptide results in release of active nisin. By targeting the universal peptidoglycan precursor lipid II, nisin has a broad target spectrum including important human pathogens such as Listeria monocytogenes and methicillin-resistant Staphylococcus aureus strains. Industrial nisin production is currently performed using natural producer strains resulting in rather low product purity and limiting its application to preservation of dairy food products.
Results
We established heterologous nisin production using the biotechnological workhorse organism Corynebacterium glutamicum in a two-step process. We demonstrate successful biosynthesis and export of fully modified prenisin and its activation to mature nisin by a purified, soluble variant of the nisin protease NisP (sNisP) produced in Escherichia coli. Active nisin was detected by a L. lactis sensor strain with strictly nisin-dependent expression of the fluorescent protein mCherry. Following activation by sNisP, supernatants of the recombinant C. glutamicum producer strain cultivated in standard batch fermentations contained at least 1.25 mg/l active nisin.
Conclusions
We demonstrate successful implementation of a two-step process for recombinant production of active nisin with C. glutamicum. This extends the spectrum of bioactive compounds that may be produced using C. glutamicum to a bacteriocin harboring complex posttranslational modifications. Our results provide a basis for further studies to optimize product yields, transfer production to sustainable substrates and purification of pharmaceutical grade nisin.
“…In silico analyses using the web-based tool BAGEL4 revealed several, yet undescribed bacteriocin gene clusters (BGC) in the genus Corynebacterium (8). In this study, we closely examined one of the predicted BGCs in the genome of C. lactis RW2-5 isolated from raw cow’s milk and found an identical cluster in the strain C. lactis RW3-42 upon sequencing of the locus (30).…”
Section: Resultsmentioning
confidence: 99%
“…Actinobacteria. Nevertheless, several studies suggest the widespread occurrence of bacteriocin gene clusters (BGCs) in non-LAB species, including the genus Corynebacterium (7)(8)(9)(10). Species of this genus are wide-spread in nature, make up one of the largest groups of bacteria in the human and animal skin microbiome and are also present in food products including raw milk or cheese (11)(12)(13).…”
Genome analysis of Corynebacterium lactis revealed a bacteriocin gene cluster encoding a putative bacteriocin of the linaridin family of ribosomally synthesized and posttranslationally modified peptides (RIPPs). The locus harbors typical linaridin modification enzymes but lacks genes for a decarboxylase and methyltransferase, which is unusual for type B linaridins. Supernatants of Corynebacterium lactis RW3-42 showed antimicrobial activity against Corynebacterium glutamicum. Deletion of the precursor gene crdA clearly linked the antimicrobial activity of the producer strain to the identified gene cluster. Following purification, we observed potent activity of the peptide against Actinobacteria, mainly other members of the genus Corynebacterium including the pathogenic species Corynebacterium striatum and Corynebacterium amycolatum. Also, low activity against some Firmicutes was observed, but no activity against Gram-negative species. The peptide is resilient towards heat but sensitive to proteolytic degradation by trypsin and proteinase K. Notably, time-kill kinetics and experiments using live biosensors to monitor membrane integrity suggest bactericidal activity that does not involve formation of pores in the cytoplasmic membrane. As Corynebacterium species are ubiquitous in nature and include important commensal and pathogens of mammalian organisms, secretion of bacteriocins by species of this genus could be a hitherto neglected trait with high relevance for intra- and interspecies competition and infection.ImportanceBacteriocins are antimicrobial peptides produced by bacteria to fend off competitors in ecological niches and are considered to be important factors influencing the composition of microbial communities. However, bacteriocin production by bacteria of the genus Corynebacterium has been a hitherto neglected trait, although its species are ubiquitous in nature and make up large parts of the microbiome of humans and animals. In this study, we describe and characterize a novel linaridin-family bacteriocin from Corynebacterium lactis and show its narrow spectrum activity, mainly against other actinobacteria. Moreover, we were able to extend the limited knowledge on linaridin bioactivity in general and for the first time describe the mode of action of such a bacteriocin. Interestingly, the peptide, which was named corynaridin, appears bactericidal, but without formation of pores in the bacterial membrane.
“…Auch hier profi tiert die Forschung der letzten Jahre jedoch von den oben beschriebenen, neuen Methoden zur Identifi zierung neuartiger Bacteriocine. Durch in silico-Analysen öffentlich zugänglicher Corynebacte rium-Genome konnten wir zeigen, dass mindestens ein Viertel der analysierten Spezies ein oder mehrere Gencluster für die Biosynthese von Bacteriocinen besitzen [7]. In den vorhergesagten Genclustern konnten indikative Gene für alle bislang beschriebenen Bacteriocin-Klassen gefunden werden, was auf ein ähnlich diversifi ziertes Vorkommen schließen lässt, wie es auch bei den Milchsäurebakterien der Fall ist.…”
Section: Bacteriocine Von Corynebakterienunclassified
Bacteriocins are gene-encoded antimicrobial peptides produced naturally by a wide range of bacteria. Their biological role is to provide producers with a competitive advantage over other bacteria in complex and densely populated habitats. Due to the dramatic increase in antibiotic resistances of many important pathogenic bacteria, bacteriocins are also discussed as potential alternatives to antibiotics. Here we discuss the potential of the members of the Corynebacteriaceae as a source for novel bacteriocins and as hosts for biotechnological production.
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