Time-resolved cell-to-cell heterogeneity of Listeria innocua after nisin exposure

The use of bacteriocins is a promising approach for addressing the immense threat of food-borne and drug-resistant pathogens. In recent years screening platforms for novel bacteriocins using whole-cell biosensors have been established. During screening cell-to-cell heterogeneity is currently neglected but might play a crucial role in signal development of the whole-cell biosensor after bacteriocin exposure. In this study, we explored the temporal dynamics of the signal heterogeneity of the biosensor Listeria innocua LMG2785/pNZpHin2Lm after nisin exposure using microfluidic single-cell analysis. The results provided novel and detailed insights into the dynamics of cell-to-cell heterogeneity in L. innocua LMG2785/pNZpHin2Lm at different nisin concentrations with a high spatio-temporal resolution. Furthermore, the formation of subpopulations during bacteriocin exposure was observed. In-depth single-cell tracking even revealed the regeneration of disrupted cells and recovery of pH homeostasis in rare instances. These findings are highly important for the future design and execution of bacteriocin assays and for the interpretation of fluorescence signal development at the population level after exposure to different concentrations of bacteriocins (here, nisin), as well as for obtaining deeper insights into single-cell persistence strategies to quantify the efficacy and efficiency of novel bacteriocins

High-efficiency production of the antimicrobial peptide pediocin PA-1 in metabolically engineered Corynebacterium glutamicum using a microaerobic process at acidic pH and elevated levels of bivalent calcium ions

Background Pediocin PA-1 is a bacteriocin of recognized value with applications in food bio-preservation and the medical sector for the prevention of infection. To date, industrial manufacturing of pediocin PA-1 is limited by high cost and low-performance. The recent establishment of the biotechnological workhorse Corynebacterium glutamicum as recombinant host for pediocin PA-1 synthesis displays a promising starting point towards more efcient production. Results Here, we optimized the fermentative production process. Following successful simplifcation of the production medium, we carefully investigated the impact of dissolved oxygen, pH value, and the presence of bivalent calcium ions on pediocin production. It turned out that the formation of the peptide was strongly supported by an acidic pH of 5.7 and microaerobic conditions at a dissolved oxygen level of 2.5%. Furthermore, elevated levels of CaCl2 boosted production. The IPTG-inducible producer C. glutamicum CR099 pXMJ19 Ptac pedACDCg provided 66 mg L−1 of pediocin PA-1 in a two-phase batch process using the optimized set-up. In addition, the novel constitutive strain Ptuf pedACDCg allowed successful production without the need for IPTG. Conclusions The achieved pediocin titer surpasses previous eforts in various microbes up to almost seven-fold, providing a valuable step to further explore and develop this important bacteriocin. In addition to its high biosynthetic performance C. glutamicum proved to be highly robust under the demanding producing conditions, suggesting its further use as host for bacteriocin production

Characterization and recombinant production of the bacteriocin garvicin Q

Bacteriocins are ribosomally synthesized peptides or small proteins, which inhibit growth or kill susceptible bacteria. These natural compounds are produced by a wide variety of bacteria and are interesting candidates to replace conventional antibiotics. So far, applications of bacteriocins focus mostly on the preservation of food and animal feed and are limited by the relatively high production costs. The focus of this doctoral thesis was to characterize the biosynthesis and mode of action of the broad-spectrum bacteriocin garvicin Q and to develop a sufficient recombinant production of garvicin Q, using the biotechnological workhorse Corynebacterium glutamicum as a host. Genome sequencing and analysis of the fermented balsam pear isolate Lactococcus petauri B1726 revealed the garvicin Q biosynthetic gene cluster. The gene garQ encodes the garvicin Q pre-peptide, garI encodes an immunity protein, garC encodes an ABC-transporter with peptidase domain and garD codes for an accessory secretion protein. GarC and presumably also GarD make up the garvicin Q transporter that is responsible for translocation of garvicin Q and processing of the precursor peptide by the conserved peptidase domain of GarC. The correctly processed mature garvicin Q was purified from L. petauri B1726 supernatants by a combination of hydrophobic interaction chromatography and reverse-phase chromatography and was subsequently identified by mass spectrometry analysis. The knowledge about the mode of action of garvicin Q was extended by applying pHluorin-based biosensor strains. The fluorescence protein pHluorin is a GFP-derivate that responds in a ratiometric manner to pH changes and can be used to indirectly detect membrane damage caused by bacteriocins like pediocin PA-1 or nisin. Aside of characterization of garvicin Q, the pHluorin assay was applied to identify membrane damaging peptides from a collection of bacterial isolates from raw cow´s milk. Based on the here presented studies the bactericidal mode of action of garvicin Q includes: (I) binding of the peptide in an unspecific manner to cell envelopes of Gram-positive bacteria, (II) interaction with transmembrane components of the mannose phosphotransferase system and (III) disruption of the membrane integrity and pH homeostasis. The production of garvicin Q with L. petauri B1726 was improved by optimizing the cultivation conditions like aeration, temperature and the medium composition to up to 200 μg ml-1. However, large-scale production of garvicin Q with the natural producer is not attractive due to its pathogenic potential for fish and humans. To overcome this limitation, recombinant production of garvicin Q with C. glutamicum was established. C. glutamicum has the advantage that its products have the GRAS (generall recognized as safe) status and in recent years’ recombinant production and secretion of other bacteriocins (pediocin PA-1 and nisin) has been established. The expression of the codon-optimized garQICD genes in C. glutamicum resulted in the production and secretion of bioactive garvicin Q and correct peptide processing was demonstrated by mass spectrometry analysis. However, the production of garvicin Q was approximately 100-fold lower when compared to the natural producer. As an alternative route for export of the recombinantly produced bacteriocin, the general secretory (Sec) pathway of C. glutamicum was employed. For this purpose, the signal peptide of the garvicin Q pre-peptide was exchanged by a variety of corynebacterial Sec signal peptides. In total, a strain library of 23 different signal peptide-garvicin Q fusions was constructed and the signal peptide leading to the highest secreted garvicin Q production was selected for further experiments. Correct processing of the peptide was again confirmed by mass spectrometry analysis. The garvicin Q production by the “Sec secretion strain” was 3.5-fold higher when compared to the C. glutamicum strain expressing garQICD, highlighting the potential of using the Secpathway for peptide production. Another bottleneck for the recombinant production of bacteriocins is the strong adsorption of respective hydrophobic and cationic peptides to the negatively charged cell envelope of bacteria. The adsorption of garvicin Q to C. glutamicum cells was reduced from ~90 % to ~20 % by lowering the extracellular pH or to even ~10 % by the addition of tween 80 and CaCl2 in in vitro assays. The addition of these supplements to C. glutamicum minimal medium facilitated the release of garvicin Q into the culture supernatant. Using a HtrA protease deficient strain, the production of garvicin Q was further increased to up to 40 μg ml-1. The stability and yield of garvicin Q was further enhanced by using non-baffled flasks instead of baffled flasks for cultivation. Combining the supplementation with tween 80 and CaCl2, the HtrA-deficient C. glutamicum strain and the cultivation in non-baffled flasks, approximately 100 μg garvicin Q ml-1 was produced. Based on the key findings for the recombinant production of garvicin Q, the recombinant production of pediocin PA-1 with C. glutamicum was also optimized. The combination of acidic extracellular pH and CaCl2 helped to markedly increase the production of pediocin PA-1 from 10 μg ml-1 to up to 66 μg ml-1. This highlights, that bottlenecks observed during recombinant garvicin Q production also apply for production of other bacteriocins and that the optimized production strategy might be utilized for other interesting peptides in the future

Time-resolved cell-to-cell heterogeneity of Listeria innocua after nisin exposure

Fante N, Desiderato CK, Riedel CU, Grünberger A. Time-resolved cell-to-cell heterogeneity of Listeria innocua after nisin exposure. Frontiers in Bioengineering and Biotechnology. 2024;12:14 Seiten.The use of bacteriocins is a promising approach for addressing the immense threat of food-borne and drug-resistant pathogens. In recent years screening platforms for novel bacteriocins using whole-cell biosensors have been established. During screening cell-to-cell heterogeneity is currently neglected but might play a crucial role in signal development of the whole-cell biosensor after bacteriocin exposure. In this study, we explored the temporal dynamics of the signal heterogeneity of the biosensorListeria innocuaLMG2785/pNZpHin2Lmafter nisin exposure using microfluidic single-cell analysis. The results provided novel and detailed insights into the dynamics of cell-to-cell heterogeneity inL. innocuaLMG2785/pNZpHin2Lmat different nisin concentrations with a high spatio-temporal resolution. Furthermore, the formation of subpopulations during bacteriocin exposure was observed. In-depth single-cell tracking even revealed the regeneration of disrupted cells and recovery of pH homeostasis in rare instances. These findings are highly important for the future design and execution of bacteriocin assays and for the interpretation of fluorescence signal development at the population level after exposure to different concentrations of bacteriocins (here, nisin), as well as for obtaining deeper insights into single-cell persistence strategies to quantify the efficacy and efficiency of novel bacteriocins

Homologe und heterologe Produktion von Bacteriocinen mit Corynebakterien

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

Identification of Potential Probiotics Producing Bacteriocins Active against Listeria monocytogenes by a Combination of Screening Tools

Listeria monocytogenes is an important food-borne pathogen and a serious concern to food industries. Bacteriocins are antimicrobial peptides produced naturally by a wide range of bacteria mostly belonging to the group of lactic acid bacteria (LAB), which also comprises many strains used as starter cultures or probiotic supplements. Consequently, multifunctional strains that produce bacteriocins are an attractive approach to combine a green-label approach for food preservation with an important probiotic trait. Here, a collection of bacterial isolates from raw cow’s milk was typed by 16S rRNA gene sequencing and MALDI-Biotyping and supernatants were screened for the production of antimicrobial compounds. Screening was performed with live Listeria monocytogenes biosensors using a growth-dependent assay and pHluorin, a pH-dependent protein reporting membrane damage. Purification by cation exchange chromatography and further investigation of the active compounds in supernatants of two isolates belonging to the species Pediococcus acidilactici and Lactococcus garvieae suggest that their antimicrobial activity is related to heat-stable proteins/peptides that presumably belong to the class IIa bacteriocins. In conclusion, we present a pipeline of methods for high-throughput screening of strain libraries for potential starter cultures and probiotics producing antimicrobial compounds and their identification and analysis

Garvicin Q: characterization of biosynthesis and mode of action

Bacteriocins are ribosomally synthesized antimicrobial peptides, that either kill target bacteria or inhibit their growth. Bacteriocins are used in food preservation and are of increasing interest as potential alternatives to conventional antibiotics. In the present study, we show that Lactococcus petauri B1726, a strain isolated from fermented balsam pear, produces a heat-stable and protease-sensitive compound. Following genome sequencing, a gene cluster for production of a class IId bacteriocin was identified consisting of garQ (encoding for the bacteriocin garvicin Q), garI (for a putative immunity protein), garC, and garD (putative transporter proteins). Growth conditions were optimized for increased bacteriocin activity in supernatants of L. petauri B1726 and purification and mass spectrometry identified the compound as garvicin Q. Further experiments suggest that garvicin Q adsorbs to biomass of various susceptible and insusceptible bacteria and support the hypothesis that garvicin Q requires a mannose-family phosphotransferase system (PTS(Man)) as receptor to kill target bacteria by disruption of membrane integrity. Heterologous expression of a synthetic garQICD operon was established in Corynebacterium glutamicum demonstrating that genes garQICD are responsible for biosynthesis and secretion of garvicin Q. Moreover, production of garvicin Q by the recombinant C. glutamicum strain was improved by using a defined medium yet product levels were still considerably lower than with the natural L. petauri B1726 producer strain. Collectively, our data identifies the genetic basis for production of the bacteriocin garvicin Q by L. petauri B1726 and provides insights into the receptor and mode of action of garvicin Q. Moreover, we successfully performed first attempts towards biotechnological production of this interesting bacteriocin using natural and heterologous hosts. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-022-01952-9

Garvicin Q-Characterization of biosynthesis and mode of action

Bacteriocins are ribosomally synthesized antimicrobial peptides, that either kill target bacteria or inhibit their growth. Bacteriocins are used in food preservation and are of increasing interest as potential alternatives to conventional antibiotics. In the present study, we show that Lactococcus petauri B1726, a strain isolated from fermented balsam pear, produces a heat-stable and protease-sensitive compound. Following genome sequencing, a gene cluster for production of a class IId bacteriocin was identified consisting of garQ (encoding for the bacteriocin garvicin Q), garI (for a putative immunity protein), garC, and garD (putative transporter proteins). Growth conditions were optimized for increased bacteriocin activity in supernatants of L. petauri B1726 and purification and mass spectrometry identified the compound as garvicin Q. Further experiments suggest that garvicin Q adsorbs to biomass of various susceptible and insusceptible bacteria and support the hypothesis that garvicin Q requires a mannose-family phosphotransferase system (PTS(Man)) as receptor to kill target bacteria by disruption of membrane integrity. Heterologous expression of a synthetic garQICD operon was established in Corynebacterium glutamicum demonstrating that genes garQICD are responsible for biosynthesis and secretion of garvicin Q. Moreover, production of garvicin Q by the recombinant C. glutamicum strain was improved by using a defined medium yet product levels were still considerably lower than with the natural L. petauri B1726 producer strain. Collectively, our data identifies the genetic basis for production of the bacteriocin garvicin Q by L. petauri B1726 and provides insights into the receptor and mode of action of garvicin Q. Moreover, we successfully performed first attempts towards biotechnological production of this interesting bacteriocin using natural and heterologous hosts. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-022-01952-9

Data_Sheet_1_Optimized recombinant production of the bacteriocin garvicin Q by Corynebacterium glutamicum.PDF

Bacteriocins are antimicrobial peptides applied in food preservation and are interesting candidates as alternatives to conventional antibiotics or as microbiome modulators. Recently, we established Corynebacterium glutamicum as a suitable production host for various bacteriocins including garvicin Q (GarQ). Here, we establish secretion of GarQ by C. glutamicum via the Sec translocon achieving GarQ titers of about 7 mg L–1 in initial fermentations. At neutral pH, the cationic peptide is efficiently adsorbed to the negatively charged envelope of producer bacteria limiting availability of the bacteriocin in culture supernatants. A combination of CaCl2 and Tween 80 efficiently reduces GarQ adsorption to C. glutamicum. Moreover, cultivation in minimal medium supplemented with CaCl2 and Tween 80 improves GarQ production by C. glutamicum to about 15 mg L–1 but Tween 80 resulted in reduced GarQ activity at later timepoints. Using a reporter strain and proteomic analyses, we identified HtrA, a protease associated with secretion stress, as another potential factor limiting GarQ production. Transferring production to HtrA-deficient C. glutamicum K9 improves GarQ titers to close to 40 mg L–1. Applying conditions of low aeration prevented loss in activity at later timepoints and improved GarQ titers to about 100 mg L–1. This is about 50-fold higher than previously shown with a C. glutamicum strain employing the native GarQ transporter GarCD for secretion and in the range of levels observed with the native producer Lactococcus petauri B1726. Additionally, we tested several synthetic variants of GarQ and were able to show that exchange of the methionine in position 5 to a phenylalanine (GarQM5F) results in markedly increased activity against Lactococcus lactis and Listeria monocytogenes. In summary, our findings shed light on several aspects of recombinant GarQ production that may also be of relevance for production with natural producers and other bacteriocins.</p