The zeamine antibiotics affect the integrity of bacterial membranes

The zeamines (zeamine, zeamine I, and zeamine II) constitute an unusual class of cationic polyamine-polyketide-nonribosomal peptide antibiotics produced by Serratia plymuthica RVH1. They exhibit potent bactericidal activity, killing a broad range of Gram-negative and Gram-positive bacteria, including multidrug-resistant pathogens. Examination of their specific mode of action and molecular target revealed that the zeamines affect the integrity of cell membranes. The zeamines provoke rapid release of carboxyfluorescein from unilamellar vesicles with different phospholipid compositions, demonstrating that they can interact directly with the lipid bilayer in the absence of a specific target. DNA, RNA, fatty acid, and protein biosynthetic processes ceased simultaneously at subinhibitory levels of the antibiotics, presumably as a direct consequence of membrane disruption. The zeamine antibiotics also facilitated the uptake of small molecules, such as 1-N-phenylnaphtylamine, indicating their ability to permeabilize the Gram-negative outer membrane (OM). The valine-linked polyketide moiety present in zeamine and zeamine I was found to increase the efficiency of this process. In contrast, translocation of the large hydrophilic fluorescent peptidoglycan binding protein PBDKZ-GFP was not facilitated, suggesting that the zeamines cause subtle perturbation of theOMrather than drastic alterations or defined pore formation. At zeamine concentrations above those required for growth inhibition, membrane lysis occurred as indicated by time-lapse microscopy. Together, these findings show that the bactericidal activity of the zeamines derives from generalized membrane permeabilization, which likely is initiated by electrostatic interactions with negatively charged membrane components

The orphan germinant receptor protein GerXAO (but not GerX3b) is essential for L-alanine induced germination in Clostridium botulinum Group II

Clostridium botulinum is an anaerobic spore forming bacterium that produces the potent botulinum neurotoxin that causes a severe and fatal neuro-paralytic disease of humans and animals (botulism). C. botulinum Group II is a psychrotrophic saccharolytic bacterium that forms spores of moderate heat resistance and is a particular hazard in minimally heated chilled foods. Spore germination is a fundamental process that allows the spore to transition to a vegetative cell and typically involves a germinant receptor (GR) that responds to environmental signals. Analysis of C. botulinum Group II genomes shows they contain a single GR cluster (gerX3b), and an additional single gerA subunit (gerXAO). Spores of C. botulinum Group II strain Eklund 17B germinated in response to the addition of L-alanine, but did not germinate following the addition of exogenous Ca2+-DPA. Insertional inactivation experiments in this strain unexpectedly revealed that the orphan GR GerXAO is essential for L-alanine stimulated germination. GerX3bA and GerX3bC affected the germination rate but were unable to induce germination in the absence of GerXAO. No role could be identified for GerX3bB. This is the first study to identify the functional germination receptor of C. botulinum Group II

Insight into the germination mechanism of psychrotrophic group II Clostridium botulinum (type E) spores

Clostridium botulinum is a strictly anaerobic spore-forming bacterium, and produces the most deadly toxin known, the botulinum neurotoxin (BoNT). Therefore, food contaminated with (spores of) this pathogen constitutes an important threat to humans and animals. Bacterial spores are highly resistant dormant structures that are resilient against many hostile conditions like heat, dryness, lack of nutrients, UV, chemicals and oxygen. When conditions become favorable, they can grow out and resume vegetative growth, which is accompanied with toxin production. Since spores first have to complete a complex cascade of events designated as germination, before they can grow out, outgrowth and toxin production can in principle be prevented by inhibition of germination. However, the germination mechanism of C. botulinum spores has only been unraveled in very little detail, mainly due to biosafety and bioterrorism restrictions and because a strict anaerobic environment is required to grow the pathogen. In this PhD thesis, we focused on psychrotrophic group II C. botulinum (gIICb), the major pathogen of concern in minimally processed chilled ready-to-eat foods with extended shelf life. Due to biosafety reasons, nontoxigenic mutants were first constructed to facilitate the research on the germination mechanism. To this end, a novel deletion strategy was developed and used to create a ∆bontE mutant in C. botulinum NCTC 11219. This was the first report of a deletion mutant in gIICb, a species group that is known to be less genetically tractable than other clostridia, and for which the genetic toolbox is still very limited. In addition, a bontE insertional knockout mutant was created by the ClosTron system. While most characteristics of the mutants showed to be identical to the parental strain, also some differences were noted. Presumably, these changes are due to some spontaneous mutations that were identified by whole genome sequence analysis. It could be excluded that the changed properties are associated with BoNT inactivation, since the same atoxigenic mutants were constructed (by insertion and deletion) in a different parental strain (NCTC 8266) and these did not show any altered properties, except for a growth defect at lowered temperatures for the ClosTron mutant. The deletion mutants of these two strains offer safe surrogate organisms for gIICb in challenge testing of foods. In a second part of this PhD research, the spore germination process was investigated. First, the ability to induce germination of the nutrient mixture L-alanine/L-lactate/NaHCO3, high pressure treatment, calcium dipicolinic acid (Ca2+-DPA) and dodecylamine was assessed. Based on the loss of heat resistance and the release of DPA, it was concluded that only the nutrient mixture and the surfactant dodecylamine could induce germination. However, dodecylamine germinated spores did not loose their refractility in phase-contrast microscopy, indicating that they had not rehydrated. In addition, these spores only became heat sensitive when dodecylamine was present, whereas they remained resistant when dodecylamine was washed away before the heat treatment. This suggests that dodecylamine does not induce genuine germination. Furthermore, four natural antimicrobial compounds (carvacrol, cinnamaldehyde, carrot seed essential oil and hop β-acids) were tested for their inhibitory action on germination. Although these compounds have mainly been studied for their growth inhibiting action, the inhibition of spore germination could also contribute to the control of spore-formers in foods. The results demonstrated that the minimal concentration of carvacrol, cinnamaldehyde and carrot seed essential oil that could significantly reduce nutrient germination, was many times lower than the minimal inhibitory concentration (MIC) on vegetative growth. This could be of interest because the low sensory- and odor thresholds often interfere with the application of these compounds in foods. In a more mechanistically oriented part, specific genes that are predicted to encode proteins involved in germination of gIICb, were investigated via deletion analysis. The presence of only one Ger-type germinant receptor, GerX3b, in these strains is unusual because other spore-formers generally contain more receptors. Moreover it is intriguing, because it is unclear how a single receptor can respond to the many different nutrients that can induce germination in gIICb. The gerBAC gene cluster, encoding the three subunits of the GerX3b receptor, was successfully deleted. Germination experiments with this mutant showed that the GerX3b receptor is not involved in germination induced by different nutrient mixtures. Furthermore, two predicted cortex hydrolases, SleB and SleC, were investigated via the same knockout strategy, but a deletion mutant could only be successfully created for SleB. Because spores lacking SleB could still complete cortex hydrolysis, it was concluded that SleB is not an essential cortex hydrolase in gIICb, similar to what has been reported for SleB of C. difficile. Nevertheless, an auxiliary role in other germination events can not be excluded since the germination assays showed a large variability.status: publishe

Construction of nontoxigenic mutants of nonproteolytic Clostridium botulinum NCTC 11219 by insertional mutagenesis and gene replacement

UNLABELLED: Group II nonproteolytic Clostridium botulinum (gIICb) strains are an important concern for the safety of minimally processed ready-to-eat foods, because they can grow and produce botulinum neurotoxin during refrigerated storage. The principles of control of gIICb by conventional food processing and preservation methods have been well investigated and translated into guidelines for the food industry; in contrast, the effectiveness of emerging processing and preservation techniques has been poorly documented. The reason is that experimental studies with C. botulinum are cumbersome because of biosafety and biosecurity concerns. In the present work, we report the construction of two nontoxigenic derivatives of the type E gIICb strain NCTC 11219. In the first strain, the botulinum toxin gene (bont/E) was insertionally inactivated with a retargeted intron using the ClosTron system. In the second strain, bont/E was exchanged for an erythromycin resistance gene using a new gene replacement strategy that makes use of pyrE as a bidirectional selection marker. Growth under optimal and stressed conditions, sporulation efficiency, and spore heat resistance of the mutants were unaltered, except for small differences in spore heat resistance at 70°C and in growth at 2.3% NaCl. The mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with gIICb. In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in gIICb and other clostridia. IMPORTANCE: The nontoxigenic mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with psychrotrophic Clostridium botulinum In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in clostridia.status: publishe

Two Complete and One Draft Genome Sequence of Nonproteolytic Clostridium botulinum Type E Strains NCTC 8266, NCTC 8550, and NCTC 11219

Group II (gII) nonproteolytic Clostridium botulinum strains are a major cause of foodborne botulism outbreaks. Here, we report two complete genome sequences of gII type E strains NCTC 8266 and NCTC 8550 and one draft genome sequence of type E NCTC 11219.status: publishe

Construction of Nontoxigenic Mutants of Nonproteolytic Clostridium botulinum NCTC 11219 by Insertional Mutagenesis and Gene Replacement.

&lt;p&gt;&lt;b&gt;UNLABELLED: &lt;/b&gt;Group II nonproteolytic Clostridium botulinum (gIICb) strains are an important concern for the safety of minimally processed ready-to-eat foods, because they can grow and produce botulinum neurotoxin during refrigerated storage. The principles of control of gIICb by conventional food processing and preservation methods have been well investigated and translated into guidelines for the food industry; in contrast, the effectiveness of emerging processing and preservation techniques has been poorly documented. The reason is that experimental studies with C. botulinum are cumbersome because of biosafety and biosecurity concerns. In the present work, we report the construction of two nontoxigenic derivatives of the type E gIICb strain NCTC 11219. In the first strain, the botulinum toxin gene (bont/E) was insertionally inactivated with a retargeted intron using the ClosTron system. In the second strain, bont/E was exchanged for an erythromycin resistance gene using a new gene replacement strategy that makes use of pyrE as a bidirectional selection marker. Growth under optimal and stressed conditions, sporulation efficiency, and spore heat resistance of the mutants were unaltered, except for small differences in spore heat resistance at 70°C and in growth at 2.3% NaCl. The mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with gIICb. In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in gIICb and other clostridia.&lt;/p&gt;&lt;p&gt;&lt;b&gt;IMPORTANCE: &lt;/b&gt;The nontoxigenic mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with psychrotrophic Clostridium botulinum In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in clostridia.&lt;/p&gt;</p

Two complete and one draft genome sequence of nonproteolytic Clostridium botulinum type E strains NCTC 8266, NCTC 8550 and NCTC 11219

Group II (gII) nonproteolytic Clostridium botulinum strains are a major cause of foodborne botulism outbreaks. Here, we report two complete genome sequences of gII type E strains NCTC 8266 and NCTC 8550 and one draft genome sequence of type E NCTC 11219