Aneuploidy Formation in the Filamentous Fungus Aspergillus flavus in Response to Azole Stress.

Aspergillus flavus is a mycotoxigenic fungus that contaminates many important agricultural crops with aflatoxin B1, the most toxic and carcinogenic natural compound. This fungus is also the second leading cause of human invasive aspergillosis, after Aspergillus fumigatus, a disease that is particularly prevalent in immunocompromised individuals. Azole drugs are considered the most effective compounds in controlling Aspergillus infections both in clinical and agricultural settings. Emergence of azole resistance in Aspergillus spp. is typically associated with point mutations in cyp51 orthologs that encode lanosterol 14α-demethylase, a component of the ergosterol biosynthesis pathway that is also the target of azoles. We hypothesized that alternative molecular mechanisms are also responsible for acquisition of azole resistance in filamentous fungi. We found that an aflatoxin-producing A. flavus strain adapted to voriconazole exposure at levels above the MIC through whole or segmental aneuploidy of specific chromosomes. We confirm a complete duplication of chromosome 8 in two sequentially isolated clones and a segmental duplication of chromosome 3 in another clone, emphasizing the potential diversity of aneuploidy-mediated resistance mechanisms. The plasticity of aneuploidy-mediated resistance was evidenced by the ability of voriconazole-resistant clones to revert to their original level of azole susceptibility following repeated transfers on drug-free media. This study provides new insights into mechanisms of azole resistance in a filamentous fungus. IMPORTANCE Fungal pathogens cause human disease and threaten global food security by contaminating crops with toxins (mycotoxins). Aspergillus flavus is an opportunistic mycotoxigenic fungus that causes invasive and noninvasive aspergillosis, diseases with high rates of mortality in immunocompromised individuals. Additionally, this fungus contaminates most major crops with the notorious carcinogen, aflatoxin. Voriconazole is the drug of choice to treat infections caused by Aspergillus spp. Although azole resistance mechanisms have been well characterized in clinical isolates of Aspergillus fumigatus, the molecular basis of azole resistance in A. flavus remains unclear. Whole-genome sequencing of eight voriconazole-resistant isolates revealed that, among other factors, A. flavus adapts to high concentrations of voriconazole by duplication of specific chromosomes (i.e., aneuploidy). Our discovery of aneuploidy-mediated resistance in a filamentous fungus represents a paradigm shift, as this type of resistance was previously thought to occur only in yeasts. This observation provides the first experimental evidence of aneuploidy-mediated azole resistance in the filamentous fungus A. flavus.

Ensiling process and pomegranate peel extract as a natural additive in potential prevention of fungal and mycotoxin contamination in silage.

A study was conducted on six animal feed centers in Israel where fungal and mycotoxin presence was examined in maize and wheat silages. Fumonisin mycotoxins FB1 and FB2 were present in every maize silage sample analyzed. Interestingly, no correlation was found between the occurrence of specific mycotoxins and the presence of the fungal species that might produce them in maize and wheat silages. We further investigated the effect of pomegranate peel extract (PPE) on Fusarium infection and fumonisin biosynthesis in laboratory-prepared maize silage. PPE had an inhibitory effect on FB1 and FB2 biosynthesis by Fusarium proliferatum, which resulted in up to 90 % reduction of fumonisin production in silage samples compared to untreated controls. This finding was supported by qRT-PCR analysis, showing downregulation of key genes involved in the fumonisin-biosynthesis pathway under PPE treatment. Our results present promising new options for the use of natural compounds that may help reduce fungal and mycotoxin contamination in agricultural foodstuff, and potentially replace traditionally used synthetic chemicals.

Low-Resolution Raman Spectroscopy for the detection of contaminant species in algal bioreactors.

Fouling of aquatic systems by harmful microalgal and cyanobacterial species is an environmental and public health concern. Microalgal bioreactors are engineered ecosystems for the cultivation of algal biomass to meet the increasing demand for alternative protein sources and algae-derived products. Such bioreactors are often open or semi-open ponds or raceways that are prone to contamination by contaminant photosynthetic microorganisms, including harmful cyanobacterial species (HCBs). HCBs affect the quality of products through the accumulation of off-flavours, reducing their acceptance by consumers, and through the production of several different toxins collectively known as cyanotoxins. The density of cultured species within the bioreactor environment creates difficulty in detecting low concentrations of contaminant cells, and there is currently no technology enabling rapid monitoring of contaminations. The present study demonstrates the potential of Low-Resolution Raman Spectroscopy (LRRS) as a tool for rapid detection of low concentrations of HCBs within dense populations of the spirulina (Arthrospira platensis) cultures. An LRRS system adapted for the direct measurement of raw biomass samples was used to assemble a database of Raman spectral signatures, from eight algal and cyanobacterial strains. This dataset was used to develop both quantitative and discriminative chemometric models. The results obtained from the chemometric analyses demonstrate the ability of the LRRS to detect and quantify algal and cyanobacterial species at concentrations as low as 103 cells/mL and to robustly discriminate between species at concentrations of 104 cells/mL. The LRRS and chemometric analyses were further able to detect the presence of low concentrations (103cells/mL) of contaminating species, including the toxic cyanobacterium Microcystis aeruginosa, within dense (>107 cells/mL) spirulina cultures. The results presented provide a first demonstration of the potential of LRRS technology for real-time detection of contaminant species within microalgal bioreactors, and possibly for early detection of developing harmful algal blooms in other aquatic ecosystems.

Analysis of Stored Wheat Grain-Associated Microbiota Reveals Biocontrol Activity among Microorganisms against Mycotoxigenic Fungi.

Wheat grains are colonized by complex microbial communities that have the potential to affect seed quality and susceptibility to disease. Some of the beneficial microbes in these communities have been shown to protect plants against pathogens through antagonism. We evaluated the role of the microbiome in seed health: in particular, against mycotoxin-producing fungi. Amplicon sequencing was used to characterize the seed microbiome and determine if epiphytes and endophytes differ in their fungal and bacterial diversity and community composition. We then isolated culturable fungal and bacterial species and evaluated their antagonistic activity against mycotoxigenic fungi. The most prevalent taxa were found to be shared between the epiphytic and endophytic microbiota of stored wheat seeds. Among the isolated bacteria, Bacillus strains exhibited strong antagonistic properties against fungal pathogens with noteworthy fungal load reduction in wheat grain samples of up to a 3.59 log10 CFU/g compared to untreated controls. We also found that a strain of the yeast, Rhodotorula glutinis, isolated from wheat grains, degrades and/or metabolizes aflatoxin B1, one of the most dangerous mycotoxins that negatively affects physiological processes in animals and humans. The mycotoxin level in grain samples was significantly reduced up to 65% in the presence of the yeast strain, compared to the untreated control. Our study demonstrates that stored wheat grains are a rich source of bacterial and yeast antagonists with strong inhibitory and biodegradation potential against mycotoxigenic fungi and the mycotoxins they produce, respectively. Utilization of these antagonistic microorganisms may help reduce fungal and mycotoxin contamination, and potentially replace traditionally used synthetic chemicals.

Rapid Detection and Quantification of Patulin and Citrinin Contamination in Fruits.

Patulin (PAT) and citrinin (CTN) are the most common mycotoxins produced by Penicillium and Aspergillus species and are often associated with fruits and fruit by-products. Hence, simple and reliable methods for monitoring these toxins in foodstuffs are required for regular quality assessment. In this study, we aimed to establish a cost-effective method for detection and quantification of PAT and CTN in pome fruits, such as apples and pears, using high-performance liquid chromatography (HPLC) coupled with spectroscopic detectors without the need for any clean-up steps. The method showed good performance in the analysis of these mycotoxins in apple and pear fruit samples with recovery ranges of 55-97% for PAT and 84-101% for CTN, respectively. The limits of detection (LOD) of PAT and CTN in fruits were 0.006 µg/g and 0.001 µg/g, while their limits of quantification (LOQ) were 0.018 µg/g and 0.003 µg/g, respectively. The present findings indicate that the newly developed HPLC method provides rapid and accurate detection of PAT and CTN in fruits.

Functional roles of LaeA, polyketide synthase, and glucose oxidase in the regulation of ochratoxin A biosynthesis and virulence in Aspergillus carbonarius.

Aspergillus carbonarius is the major producer of ochratoxin A (OTA) among Aspergillus species, but the contribution of this secondary metabolite to fungal virulence has not been assessed. We characterized the functions and addressed the roles of three factors in the regulation of OTA synthesis and pathogenicity in A. carbonarius: LaeA, a transcriptional factor regulating the production of secondary metabolites; polyketide synthase, required for OTA biosynthesis; and glucose oxidase (GOX), regulating gluconic acid (GLA) accumulation and acidification of the host tissue during fungal growth. Deletion of laeA in A. carbonarius resulted in significantly reduced OTA production in colonized nectarines and grapes. The ∆laeA mutant was unable to efficiently acidify the colonized tissue, as a direct result of diminished GLA production, leading to attenuated virulence in infected fruit compared to the wild type (WT). The designed Acpks-knockout mutant resulted in complete inhibition of OTA production in vitro and in colonized fruit. Interestingly, physiological analysis revealed that the colonization pattern of the ∆Acpks mutant was similar to that of the WT strain, with high production of GLA in the colonized tissue, suggesting that OTA accumulation does not contribute to A. carbonarius pathogenicity. Disruption of the Acgox gene inactivated GLA production in A. carbonarius, and this mutant showed attenuated virulence in infected fruit compared to the WT strain. These data identify the global regulator LaeA and GOX as critical factors modulating A. carbonarius pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection.

Host Factors Modulating Ochratoxin A Biosynthesis during Fruit Colonization by Aspergillus carbonarius.

Aspergillus carbonarius is a strong and consistent ochratoxin A (OTA) producer and considered to be the main source of this toxic metabolite in grapes and grape products such as wine, grape juice and dried vine fruit. OTA is produced under certain growth conditions and its accumulation is affected by several environmental factors, such as growth phase, substrate, temperature, water activity and pH. In this study, we examined the impact of fruit host factors on regulation and accumulation of OTA in colonized grape berries, and assessed in vitro the impact of those factors on the transcriptional levels of the key genes and global regulators contributing to fungal colonization and mycotoxin synthesis. We found that limited sugar content, low pH levels and high malic acid concentrations activated OTA biosynthesis by A. carbonarius, both in synthetic media and during fruit colonization, through modulation of global regulator of secondary metabolism, laeA and OTA gene cluster expression. These findings indicate that fruit host factors may have a significant impact on the capability of A. carbonarius to produce and accumulate OTA in grapes.

The pH-Responsive Transcription Factor PacC Governs Pathogenicity and Ochratoxin A Biosynthesis in Aspergillus carbonarius.

Pathogenic fungi must respond effectively to changes in environmental pH for successful host colonization, virulence and toxin production. Aspergillus carbonarius is a mycotoxigenic pathogen with the ability to colonize many plant hosts and secrete ochratoxin A (OTA). In this study, we characterized the functions and addressed the role of PacC-mediated pH signaling in A. carbonarius pathogenicity using designed pacC gene knockout mutant. ΔAcpacC mutant displayed an acidity-mimicking phenotype, which resulted in impaired fungal growth at neutral/alkaline pH, accompanied by reduced sporulation and conidial germination compared to the wild type (WT) strain. The ΔAcpacC mutant was unable to efficiently acidify the growth media as a direct result of diminished gluconic and citric acid production. Furthermore, loss of AcpacC resulted in a complete inhibition of OTA production at pH 7.0. Additionally, ΔAcpacC mutant exhibited attenuated virulence compared to the WT toward grapes and nectarine fruits. Reintroduction of pacC gene into ΔAcpacC mutant restored the WT phenotype. Our results demonstrate important roles of PacC of A. carbonarius in OTA biosynthesis and in pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection.

Synergistic Inhibition of Mycotoxigenic Fungi and Mycotoxin Production by Combination of Pomegranate Peel Extract and Azole Fungicide.

Fungal plant pathogens cause considerable losses in yield and quality of field crops worldwide. In addition, under specific environmental conditions, many fungi, including such as some Fusarium and Aspergillus spp., are further able to produce mycotoxins while colonizing their host, which accumulate in human and animal tissues, posing a serious threat to consumer health. Extensive use of azole fungicides in crop protection stimulated the emergence of acquired azole resistance in some plant and human fungal pathogens. Combination treatments, which become popular in clinical practice, offer an alternative strategy for managing potentially resistant toxigenic fungi and reducing the required dosage of specific drugs. In the current study we tested the effect of pomegranate peel extract (PPE) on the growth and toxin production of the mycotoxigenic fungi Aspergillus flavus and Fusarium proliferatum, both alone and in combination with the azole fungicide prochloraz (PRZ). Using time-lapse microscopy and quantitative image analysis we demonstrate significant delay of conidial germination and hyphal elongation rate in both fungi following PPE treatment in combination with PRZ. Moreover, PPE treatment reduced aflatoxin production by A. flavus up to 97%, while a combined treatment with sub-inhibitory doses of PPE and PRZ resulted in complete inhibition of toxin production over a 72 h treatment. These findings were supported by qRT-PCR analysis, showing down-regulation of key genes involved in the aflatoxin biosynthetic pathway under combined PPE/PRZ treatment al low concentrations. Our results provide first evidence for synergistic effects between the commercial drug PRZ and natural compound PPE. Future application of these findings may allow to reduce the required dosage of PRZ, and possibly additional azole drugs, to inhibit mycotoxigenic fungi, ultimately reducing potential concerns over exposure to high doses of these potentially harmful fungicides.

N-Halamine Derivatized Nanoparticles with Selective Cyanocidal Activity: Potential for Targeted Elimination of Harmful Cyanobacterial Blooms.

Harmful cyanobacterial blooms (HCBs) are becoming a major challenge for the management of both natural and man-made freshwater lakes and reservoirs. Phytoplankton communities are an essential component of aquatic ecosystems, providing the basis for natural food webs as well as important environmental services. HCBs, driven by a combination of environmental pollution and rising global temperatures, destabilize phytoplankton communities with major impacts on aquatic ecology and trophic interactions. Application of currently available algaecides generally results in unselective elimination of phytoplankton species, disrupting water ecology and environmental services provided by beneficial algae. There is thus a need for selective cyanocidal compounds that can eliminate cyanobacteria while preserving algal members of the phytoplankton community. Here, we demonstrate the efficacy of N-halamine derivatized nanoparticles (Cl NPs) in selectively eliminating cyanobacteria, including the universal bloom-forming species Microcystis aeruginosa, while having minimal effect on co-occurring algal species. We further support these results with the use a simple microfluidic platform in combination with advanced live-imaging microscopy to study the effects of Cl NPs on both laboratory cultures and natural populations of cyanobacteria and algae at single cell resolutions. We note that the Cl NPs used in this work were made of polymethacrylamide, a nonbiodegradable polymer that may be unsuitable for use as a cyanocide in open aquatic environments. Nevertheless, the demonstrated selective action of these Cl NPs suggests a potential for developing alternative, biodegradable carriers with similar properties as future cyanocidal agents that will enable selective elimination of HCBs.

Shifts in the Composition of the Microbiota of Stored Wheat Grains in Response to Fumigation.

While the wheat-associated microbiome is of major agricultural importance, little is known about the alterations in wheat grain microbial community composition during storage. Characterization of the bacterial and fungal communities in stored wheat grains revealed the impact of phosphine fumigation, one of the most effective methods to eliminate insects in stored commodities, on the composition of the wheat grain microbiome. High-throughput amplicon sequencing of the bacterial 16S rRNA gene and fungal internal transcribed spacer (ITS) region was used to analyze the wheat grain microbiome at different times over as 6 months period of storage. Higher bacterial diversity was found across the samples during the first (immediately after harvest) and second (3 months later) time points, with a predominance of Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Planctomycetes. A two-fold decrease in the number of bacterial operational taxonomic units (OTUs) was observed in wheat grains at the last time point (6 months later), following phosphine treatment. In contrast to the effect of phosphine on bacteria, it did not affect fungal diversity in stored grains. The majority of fungal sequences were assigned to Ascomycota, followed by Basidiomycota, Glomeromycota, and unidentified fungi, which were evenly distributed throughout the storage period. Alpha and beta diversity analyses were confirmed by examination of the cultured microbial taxa obtained from the stored wheat grains. Mycotoxin analysis of wheat grains collected after phosphine fumigation revealed the presence of Fusarium toxins, primarily deoxynivalenol (DON). Several mycotoxigenic Fusarium spp. were also detected in the same samples. Results of the present study indicate that microbiome of stored, whole wheat grains was strongly affected by phosphine fumigation, which changed the structure of the microbial community leading to shifts in species composition toward mycotoxigenic strains. A better understanding of the complex interactions within the microbial communities of stored grains will assist in the development of novel biocontrol strategies to overcome mycotoxin contamination.

Rapid Detection and Identification of Mycotoxigenic Fungi and Mycotoxins in Stored Wheat Grain.

This study aimed to assess the occurrence of toxigenic fungi and mycotoxin contamination in stored wheat grains by using advanced molecular and analytical techniques. A multiplex polymerase chain reaction (PCR) strategy was established for rapid identification of mycotoxigenic fungi, and an improved analytical method was developed for simultaneous multi-mycotoxin determination in wheat grains by liquid chromatography-tandem mass spectrometry (LC/MS/MS) without the need for any clean-up. The optimized multiplex PCR method was highly specific in detecting fungal species containing species-specific and mycotoxin metabolic pathway genes. The method was applied for evaluation of 34 wheat grain samples collected from storage warehouses for the presence of mycotoxin-producing fungi, and a few samples were found positive for Fusarium and Aspergillus species. Further chemical analysis revealed that 17 samples contained mycotoxins above the level of detection, but only six samples were found to be contaminated over the EU regulatory limits with at least one mycotoxin. Aflatoxin B1, fumonisins, and deoxynivalenol were the most common toxins found in these samples. The results showed a strong correlation between the presence of mycotoxin biosynthesis genes as analyzed by multiplex PCR and mycotoxin detection by LC/MS/MS. The present findings indicate that a combined approach might provide rapid, accurate, and sensitive detection of mycotoxigenic species and mycotoxins in wheat grains.

Bioinspired passive anti-biofouling surfaces preventing biofilm formation.

Biofilm formation enables bacteria to grow under unfavorable conditions, provides them with protection, and increases their resistance to antimicrobial agents. Once a biofilm has formed, it is difficult, and in some systems, impossible to treat. Strategies based on the release of biocidal agents have shown only transient efficiency. Herein, we present a novel bioinspired passive approach to the prevention of surface biofilm attachment by exploiting superhydrophobic surfaces formed via the self-assembly of paraffin or fluorinated wax crystals. Our surfaces show exceptional ability to inhibit biofilm formation of both Gram-positive Bacillus cereus and Gram-negative Pseudomonas aeruginosa over a 7 day period (up to 99.9% inhibition).

External pH is a cue for the behavioral switch that determines surface motility and biofilm formation of Alicyclobacillus acidoterrestris.

Bacteria use different strategies to survive unfavorable environmental conditions. Alicyclobacillus acidoterrestris is a bacterium capable of surviving extremely harsh conditions, for instance, during industrial food processing. A. acidoterrestris is a spore-forming, thermoacidophilic, nonpathogenic bacterium that commonly contaminates commercial pasteurized fruit juices and is, therefore, considered a major microbiological contaminant in the juice industry. The purpose of this study was to elucidate whether A. acidoterrestris is capable of multicellular behavior by testing its ability of biofilm formation and surface motility. A. acidoterrestris was found to be proficient in migration over a surface that is apparently powered by flagella. It was further shown that lowering the external pH leads to inhibition in surface motility of these bacteria. Concomitantly, the reduction in the external pH triggered biofilm formation of A. acidoterrestris cells. Thus, although no significant biofilm was formed at pH 4.5, robust cell adhesion and confluent biofilm formation was seen below the pH 3.6. These findings indicate that the reduction of external pH is an environmental cue for the behavioral switch that inhibits surface motility and triggers biofilm formation of A. acidoterrestris. Gaining insight into the multicellular behavior that facilitates A. acidoterrestris survival in food contact surfaces may contribute to the development of novel antimicrobial means to prevent cross-contamination caused by this bacterium.

Butyric acid released during milk lipolysis triggers biofilm formation of Bacillus species.

Bacillus species form biofilms within milking pipelines and on surfaces of equipment in the dairy industry which represent a continuous hygiene problem and can lead to serious economic losses due to food spoilage and equipment impairment. Although much is known about the mechanism by which the model organism Bacillus subtilis forms biofilms in laboratory mediums in vitro, little is known of how these biofilms are formed in natural environments such as milk. Besides, little is known of the signaling pathways leading to biofilm formation in other Bacillus species, such as Bacillus cereus and Bacillus licheniformis, both of which are known to contaminate milk. In this study, we report that milk triggers the formation of biofilm-related structures, termed bundles. We show this to be a conserved phenomenon among all Bacillus members tested. Moreover, we demonstrate that the tasA gene, which encodes a major portion of the matrix which holds the biofilm together, is vital for this process. Furthermore, we show that the free fatty acid (FFA) - butyric acid (BA), which is released during lipolysis of milk fat and demonstrates antimicrobial activity, is the potent trigger for biofilm bundle formation. We finally show that BA-triggered biofilm bundle formation is mediated by the histidine kinase, KinD. Taken together, these observations indicate that BA, which is a major FFA within milk triggers biofilm formation in a conserved mechanism among members of the Bacillus genus.

Draft Genome Sequence of Alicyclobacillus acidoterrestris Strain ATCC 49025.

Alicyclobacillus acidoterrestris is a spore-forming Gram-positive, thermo-acidophilic, nonpathogenic bacterium which contaminates commercial pasteurized fruit juices. The draft genome sequence for A. acidoterrestris strain ATCC 49025 is reported here, providing genetic data relevant to the successful adaptation and survival of this strain in its ecological niche.

Durable contact active antimicrobial materials formed by a one-step covalent modification of polyvinyl alcohol, cellulose and glass surfaces.

In this work we have applied a direct covalent linkage of quaternary ammonium salts (QAS) to prepare a series of contact active antimicrobial surfaces based on widely utilized materials. Formation of antimicrobial polyvinyl alcohol (PVA-QAS), cellulose (cellulose-QAS) and glass (glass-QAS) surfaces was achieved by one step synthesis with no auxiliary linkers. The X-ray photoelectron spectroscopy (XPS) revealed tridentate binding mode of the antimicrobial agent. The antimicrobial activity of the prepared materials was tested on Bacillus cereus, Alicyclobacillus acidoterrestris, Escherichia coli and Pseudomonas aeruginosa. Active site density of the modified materials was examined and found to correlate with their antimicrobial activity. Stability studies at different pH values and temperatures confirmed that the linkage of the bioactive moiety to the surface is robust and resistant to a range of pH and temperatures. Prolonged long-term effectiveness of the contact active materials was demonstrated by their repeated usage, without loss of the antimicrobial efficacy.

Scientific evidence of the therapeutic effects of dead sea treatments: a systematic review.

OBJECTIVES: The Dead Sea, the deepest and most saline lake on earth, has been known from biblical times for its healing properties. The aim of this systematic review was to present critically the level of evidence for the claims of therapeutic effects of Dead Sea treatments in several rheumatologic diseases and psoriasis as well as to review these treatments' safety. METHODS: All articles cited in MEDLINE under the query, "Dead Sea," were reviewed. RESULTS: We found bona fide evidence that Dead Sea treatments are especially effective in psoriasis due to both the special characteristics of solar ultraviolet radiation in the Dead Sea and the Dead Sea water balneotherapy. Dead Sea mud and Dead Sea balneotherapy have been found to be beneficial in rheumatologic diseases, including rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and knee osteoarthritis. In the safety analysis, we found no evidence for an increase in skin neoplasia, although skin actinic damage seems to be increased in patients treated in the Dead Sea. Dead Sea treatments do not lead to worsening of blood pressure. Substantial ingestion of Dead Sea water (generally in unusual near-drowning cases) is toxic and can result in cardiac rhythm disturbances because of electrolyte concentration abnormalities. Laboratory analysis of Dead Sea mud did not reveal mineral concentrations that could represent a health concern for their intended use. CONCLUSIONS: Dead Sea treatments are beneficial in several rheumatologic diseases and psoriasis and have a good safety profile.

Two different propionicins produced by Propionibacterium thoenii P-127.

The bacteriocin GBZ-1 was purified from the growth media of Propionibacterium thoenii P-127 and was found to have a molecular weight of 6000Da. P. thoenii P-127 also known as the producer of the bacteriocin PLG-1 (MW 10kDa). Under specific growth conditions, on semi-solid media, P. thoenii P-127 produced both PLG-1 and GBZ-1. The N-terminal of GBZ-1 was microsequenced, the gene was cloned and the DNA sequence was determined and identified. GBZ-1 is highly homologous to a protease-activated antimicrobial peptide (PAMP). In contrast to PAMP, it was purified in its active form and no protease digestion was required for its activation. The survival curve of indicator bacteria Lactobacillus delbrueckii subsp. lactic ATCC 4797 showed two phases. The fast phase of 20min was followed by a slow phase. While bacterial survival was reduced by 2logs during the fast phase, bacterial survival was reduced by additional 3logs up to 200min during the slow phase. GBZ-1 activity was affected by magnesium and its activity was completely abolished at 50mM magnesium chloride. Other divalent cations had no effect on GBZ-1 activity of GBZ-1. To the best of our knowledge this is the first report of a bacterium producing two different bacteriocins under different growth conditions.